Image analysis and measurement of biological samples

ABSTRACT

Methods, devices, apparatus, and systems are provided for image analysis. Methods of image analysis may include observation, measurement, and analysis of images of biological and other samples; devices, apparatus, and systems provided herein are useful for observation, measurement, and analysis of images of such samples. The methods, devices, apparatus, and systems disclosed herein provide advantages over other methods, devices, apparatus, and systems.

BACKGROUND

Analysis of biological samples from a subject may be important forhealth-related diagnosing, monitoring, or treating of the subject. Avariety of methods are known for the analysis of biological samples.However, in order to provide better diagnosing, monitoring, or treatingof subjects, improvements in the analysis of biological samples aredesired.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

Methods, devices, systems, and apparatuses described herein are usefulfor optical and image analysis or measurement of biological and othersamples.

Embodiments disclosed herein include sample holders suitable for holdingsamples, including biological samples, for optical examination, foroptical measurement, and for other examinations and measurements. Inembodiments, a sample holder having an optically transmissive portionand a portion configured to provide internal reflection of light withinthe sample holder is provided. In embodiments, internal reflections mayinclude partial internal reflection and may include total internalreflection of light. Incident light from an external light source, anddirected from one side of the sample holder, is effective to illuminatea sample within the sample holder from a plurality of directions. Inembodiments, an external light source disposed on one side of the sampleholder may provide epi-illumination of a sample within the sampleholder; may provide trans-illumination of a sample within the sampleholder; or may provide both epi-illumination and trans-illumination of asample within the sample holder.

Embodiments disclosed herein include systems including sample holderssuitable for holding samples. Such systems are suitable for use inexamining and measuring samples, including biological samples, by, e.g.,optical examination, optical measurement, and for other examinations andmeasurements. In embodiments, a system disclosed herein comprises asample holder having an optically transmissive portion and a portionconfigured to provide internal reflection of light within the sampleholder is provided. In embodiments, internal reflections within a sampleholder of a system disclosed herein may include partial internalreflection and may include total internal reflection of light. Systemsdisclosed herein may include light sources. Incident light from a lightsource external to a sample holder, and directed from one side of thesample holder, is effective to illuminate a sample within the sampleholder from a plurality of directions. In embodiments, a light sourcedisposed external to, and on one side of, the sample holder may provideepi-illumination of a sample within the sample holder; may providetrans-illumination of a sample within the sample holder; or may provideboth epi-illumination and trans-illumination of a sample within thesample holder. Systems disclosed herein may include a detector, ordetectors; such detectors may include optical detectors, and may includeother detectors. Such detectors are suitable for, and are configured to,make measurements of a sample and of objects and characteristics of asample and objects in a sample within a sample holder; such measurementsmay include qualitative measurements and quantitative measurements.Embodiments of systems as disclosed herein may include filters,apertures, gratings, lenses, and other optical elements. Embodiments ofsystems as disclosed herein may include mechanical apparatus forlocating, moving, and adjusting a sample holder, a light source, a lens,a filter, or other element or component of a system as disclosed herein.Embodiments of systems as disclosed herein may include components andelements for transferring, aliquotting, holding, heating, mixing,staining, conditioning, or otherwise preparing, manipulating or alteringa sample. Embodiments of systems as disclosed herein may includecomponents and elements for transporting, securing, filling, orotherwise manipulating a sample holder. Embodiments of systems asdisclosed herein may include components and elements for physicalmanipulation and treatment of a sample, and for physical manipulation ofa sample holder, where such components and elements may include, withoutlimitation, a pipette, a pump, a centrifuge, other mechanical apparatusfor moving and manipulating a sample, a sample holder, pipette tips,vessels, and reagents for use with a sample, or portion thereof.Embodiments of systems as disclosed herein may include components andelements for chemical analysis, including nucleic acid analysis, proteinanalysis, general chemistry analysis, electrochemical analysis, andother analyses of a sample or portion thereof.

Sample holders and systems disclosed herein may be used, and methodsdisclosed herein may be performed, at any location, including a clinicallaboratory, a research laboratory, a clinic, a hospital, a doctor'soffice, a point of service location, and any other suitable location.Samples held by sample holders disclosed herein, and samples examinedusing systems and methods disclosed herein, include any biologicalsample, and may be small biological samples. In embodiments, a samplemay be a small blood or urine sample, and may have a volume of less thanabout 250 μL, or less than about 150 μL, or less than about 100 μL, orless than about 50 μL, or less than about 25 μL, or less than about 15μL, or may be the same as or less than the volume of blood obtained froma finger-stick.

In one embodiment, a method for the measurement of a component ofinterest in cells of a cellular population in a sample is provided,including: a) obtaining a quantitative measurement of a marker presentin cells of the cellular population in the sample; b) based on themeasurement of part a), determining, with the aid of a computer, anapproximate amount of cells in the cellular population present in thesample; c) based on the results of part b), selecting an amount ofreagent to add to the sample, wherein the reagent binds specifically tothe component of interest in cells of the cellular population and isconfigured to be readily detectable; d) based on the results of part c),adding the selected amount of the reagent to the sample; e) assayingcells in the sample for reagent bound to the component of interest; andf) based on the amount of reagent bound to the component of interest,determining the amount of the component of interest in cells of thecellular population of the sample. In an embodiment of the method, thereagent of part c) is an antibody.

Applicants further disclose herein a method for the measurement of acomponent of interest in cells of a cellular population in a sample,comprising: a) obtaining a quantitative measurement of a marker presentin cells, or of a property of cells, of the cellular population in thesample; b) determining, with the aid of a computer, an approximateamount of cells in the cellular population present in the sample basedon the measurement of part a); c) adding an amount of a cell marker tothe sample, where the amount of said cell marker added is based on theresults of part b), and wherein the cell marker binds specifically tothe component of interest in cells of the cellular population and isconfigured to be readily detectable; d) assaying cells in the sample formarker bound to the component of interest; and e) determining the amountof the component of interest in cells of the cellular population of thesample based on the amount of marker bound to the component of interest.

In another embodiment, a method for focusing a microscope is provided,including: a) mixing a sample containing an object for microscopicanalysis with a reference particle having a known size, to generate amixture containing the sample and reference particle; b) positioning themixture of step a) into a light path of a microscope; c) exposing themixture of step a) to a light beam configured to visualize the referenceparticle; and d) focusing the microscope based on the position of thereference particle within the mixture, or based on the sharpness of theimage of the reference particle.

In yet another embodiment, provided herein is a method for identifying acell in a sample containing a plurality of cells, including: a) assayinga cell of the plurality of cells for at least one of: (i) the presenceof a cell surface antigen; (ii) the amount of a cell surface antigen; or(iii) cell size; b) assaying the cell of a) for at least one of: (i)nuclear size; or (ii) nuclear shape; and c) assaying the cell of a) andb) for quantitative cell light scatter, wherein the combination ofinformation from steps a), b) and c) is used to identify the cell in thesample containing a plurality of cells.

In yet another embodiment, provided herein is a system comprising adetector assembly for use with a sample holder that holds a sample to beexamined. In one non-limiting example, the sample holder is a cuvettethat has features or materials in it that enable the cuvette to beengaged and moved from one location to the detector assembly. In someembodiments, the detector assembly has a first surface that isconfigured to engage a surface of the sample holder in a manner suchthat the interface between the two does not create optical interferencein the optical pathway from the detector assembly to the sample in thesample holder. In one embodiment, there may be more than one location onthe detector assembly for one or more of the sample holders. Someembodiments may have the same sample holder for each of the locations.Optionally, some embodiments may have different sample holders for atleast some of the locations associated with the detector assembly.

In one embodiment described herein, a sample holder is provided hereinsuch as but not limited to a cuvette with optical properties,dimensions, materials, or physical features that allow for it to holdthe sample for analysis by the detector assembly while keeping itphysically separate from and not in direct contact with the detectorassembly. This can be particularly useful for sample fluids that containshaped members therein.

In one embodiment described herein, the detector assembly may be amulti-channel microscopy unit that is configured to detect, obtain, ormeasure the shape, and physical, optical, and biochemical properties ofa cell or cells in a sample, all in the same device. It can provide bothquantitative information, and descriptive information. One embodiment ofthe detector assembly may use multiple markers of the same color orwavelength, where the detector assembly is configured to deconvolutesignals originating from such markers in a sample (e.g., bound to cellsin a sample), allowing for a reduction in number of spectral channelsand light sources required in the assembly.

It should be understood that some embodiments herein may include asample holder such as but not limited to a cuvette with physicalfeatures in the shape of the cuvette material that increase dark fieldillumination where some features are configured to provide for lightreflectance (including, but not limited to, reflectance of light withinthe cuvette), and some features may optionally be configured formechanical support; in embodiments, some features may provide mechanicalsupport and also provide for light reflectance. In embodiments, a sampleholder is configured to provide trans-illumination of a sample byreflection of light within the sample holder. In embodiments, a sampleholder is configured to provide trans-illumination of a sample byreflection of light within the sample holder; such reflectance mayinclude partial internal reflection (PIR, also known as Fresnelreflection), and such reflectance may include total internal reflectance(TIR). In embodiments, a sample holder is configured to providetrans-illumination of a sample by reflection of light within the sampleholder, wherein the source of the reflected light is disposed on thesame side of the sample holder as the optics used to detect or measurethe light (i.e., the light source is an epi-illumination light source).

The system herein can simultaneously use both epi (direct) and trans(reflected) illumination in dark field imaging. This differs fromtraditional dark field imaging which uses either epi-illumination, ortrans-illumination, but not both types of illumination, and not bothtypes of illumination from a single source or single direction orlocation. Thus, the combination of epi- and trans-illumination disclosedherein, wherein the trans-illumination originates from the same lightsource as the epi-illumination, differs from known systems. Optionally,the use of a shaped sample holder such as the cuvette can be used toprovide the trans-illumination. In embodiments, a shaped sample holderis configured to provide trans-illumination by reflection of light. Inembodiments, a shaped sample holder is configured to providetrans-illumination by reflection of light within the sample holder. Inembodiments, one or more of the size, shape, surface, materials, orother feature of a shaped sample holder is effective to provide internalreflection of light within the shaped sample holder. In embodiments, oneor more of the size, shape, surface, materials, or other feature of ashaped sample holder is effective to provide partial internal reflection(PIR) of light within the shaped sample holder. In embodiments, one ormore of the size, shape, surface, materials, or other feature of ashaped sample holder is effective to provide total internal reflection(TIR) of light within the shaped sample holder. Optionally, theintensity of trans-illumination is non-negligible. In embodiments, ashaped sample holder may include a reflective surface effective toincrease trans-illumination light intensity. The dark field light sourcemay be a light-emitting diode (LED), laser, or other illumination sourcethat can provide the desired illumination or excitation wavelength(s).

In one embodiment, the combination of the microscope objective and lightsource such as but not limited to a ringlight (for dark fieldmicroscopy) is at a physical distance between them that enables acompact size for the detector assembly. In one embodiment, only light ata desired wavelength or within a desired range of wavelengths aredirected to the sample. In one embodiment, the light is non-polarizedlight. In another embodiment, the light is polarized light.

In yet another embodiment, information from the cytometry assay, eitherfrom the sample preparation phase or from the analysis phase, is used toguide or trigger a secondary procedure. In embodiments, such a secondaryprocedure may be to provide an alert for direct human review. Inembodiments, such a secondary procedure may be to use an estimated cellcount or other information obtained during a sample preparation step ofa procedure in order to guide the performance of an assay, where suchassay may be an assay in a later step of the procedure, or may be anassay in another procedure.

Techniques for counting cells can also provide ways to deal with sampleholders with uneven shapes or chamber surfaces. One method comprisesusing: a) a volume-metered channel technique to introduce a known volumeof a sample into an analysis area, such as a channel in the sampleholder. The method may include counting all cells in the sample holder.Since one knows the volume of sample, one also knows the concentrationof cells in volume (this may be performed in hydrophobic containers orcuvettes or sample holders with chambers with such surfaces). Anothermethod comprises: b) a ratio-based metric technique to mix sample with aknown amount of beads, which is used to calculate the concentration ofcells in the sample based on the number of beads observed.

In yet another embodiment described herein, a method is providedcomprising measuring formed blood components such as but not limited tomeasuring red blood cell (RBC) volume in a blood sample by causing theRBCs to assume substantially spherical shapes, and measuring the RBCvolume using dark field microscopy.

In yet another embodiment described herein, a method is providedcomprising measuring platelet volume. The method may include labelingplatelets with a fluorescent dye and measuring the size of the plateletsobserved; adding beads of known size to the sample; and comparing theobserved size of images of the beads to the observed images of theplatelets, using the beads as calibration to determine the size of theplatelets and to determine the platelet volume in the sample.

In yet further embodiments described herein, methods are provided fordetecting and measuring, in a sample, cell morphology; measurement ofcell numbers; detection of particles; measurement of particle numbers;detection of crystals; measurement of crystal numbers; detection of cellaggregates; measurement of numbers of cell aggregates; and otherproperties and quantities of or in a sample.

Accordingly, Applicants disclose herein:

A system for analyzing a sample, the system comprising: a sample holdercomprising a sample chamber configured to hold said sample, at least aportion of said sample holder comprising an optically transmissivematerial, said optically transmissive material comprising an opticallytransmissive surface and a reflective surface; and an illuminationsource configured to provide light that illuminates and passes throughsaid optically transmissive surface; wherein said sample holder isconfigured effective that said light from said illumination sourcesimultaneously provides both epi-illumination and trans-illumination toa sample in the sample holder, where epi-illumination comprises lighttraveling from said illumination source to said sample withoutreflection at a surface of the optically transmissive material of thesample holder, and where trans-illumination comprises light travelingwithin the optically transmissive material and to the sample followingat least one reflection from at least one surface of said opticallytransmissive material. In embodiments, a sample holder of a systemhaving the features disclosed herein may comprise a cuvette having anelongated channel configured for holding a sample. In embodiments, thesample holder may have one or more optically non-transmissive surfaces.

In embodiments of systems disclosed herein, said trans-illumination maybe provided at least in part by internal reflection of light at asurface, and may be provided at least in part by total internalreflection of light within the cuvette. In embodiments of systemsdisclosed herein, said trans-illumination may be provided at least inpart by partial internal reflection of light at a surface, and may beprovided at least in part by partial internal reflection of light withinthe cuvette.

In embodiments, a sample holder may have two or more sample chambers forholding sample. A sample holder, e.g., a cuvette, having featuredisclosed herein may have a rectangular horizontal, cross-sectionalshape; may have a circular horizontal, cross-sectional shape; may have asaw tooth vertical cross-sectional shape; may have a step-shapedvertical cross-sectional shape; or may have another shape.

In embodiments, a sample holder may be movable relative to anillumination source, and may be movable to a plurality of locations,wherein an optically transmissive surface of the sample holder may beilluminated by the illumination source at each location.

In embodiments, an illumination source may include a ringlight. Inembodiments, a ringlight may be selected from a light emitting diode(LED)-based ringlight and a laser-based ringlight.

In embodiments, a system as disclosed herein may include a supportstructure having an optically transmissive surface shaped to engage anoptically transmissive surface of the sample holder.

In embodiments, a system as disclosed herein may have a compressiondevice configured to retain the sample holder in a desired location forillumination by the illumination source.

In embodiments, a system as disclosed herein may include a detectorconfigured to image at least a portion of a channel in the sampleholder.

In embodiments, a sample holder as disclosed herein may include anelongated channel configured to contain at least a portion of thesample, and wherein a detector is configured to image an entireelongated channel in the sample holder.

In embodiments, a sample holder as disclosed herein may be configured tohold the sample in a static, non-flowing manner during imaging; inembodiments, a sample holder may be configured to hold one portion ofthe sample in a static, non-flowing manner and another portion in aflowing manner.

In embodiments, an illumination source as disclosed herein may bemovable relative to the sample holder.

In embodiments, a sample holder as disclosed herein may be configured tohold the sample in a flowing manner during imaging.

In embodiments, a sample holder as disclosed herein may include a fluidcircuit fully confined in the sample holder, and wherein the sample islocated in said fluid circuit, effective that the sample remainsseparate from said detector.

In embodiments, a sample holder as disclosed herein is movable relativeto the detector. In embodiments, a detector as disclosed herein ismovable relative to the sample holder.

In embodiments, a sample holder and an illumination source as disclosedherein comprise at least part of an optical analysis unit, and thesystem further includes a clinical analysis unit configured to performclinical analysis on a sample.

In embodiments, a system as disclosed herein is configured to provide analiquot of a single sample to an optical analysis unit and to a clinicalanalysis unit, effective that the clinical analysis unit and the opticalanalysis unit may perform optical analysis and clinical analysis onportions of a sample at the same time. In embodiments, such a clinicalanalysis may be selected from general chemical analysis, nucleic acidanalysis, and enzyme-linked binding analysis.

In embodiments, a system as disclosed herein may include a plurality ofclinical analysis units, wherein each of such clinical analysis units isconfigured to provide a clinical analysis selected from general chemicalanalysis, nucleic acid analysis, and enzyme-linked binding analysis.

Applicants further provide a cuvette comprising a sample chamberconfigured to hold a sample, at least a portion of said cuvettecomprising an optically transmissive material, said opticallytransmissive material comprising an optically transmissive surface and areflective surface, wherein said optically transmissive surface and saidreflective surface are configured effective that light passing throughthe optically transmissive surface simultaneously provides bothepi-illumination and trans-illumination to said sample in the samplechamber, where epi-illumination comprises light traveling from saidillumination source to the sample without reflection at a surface of theoptically transmissive material, and where trans-illumination compriseslight traveling within the optically transmissive material and to thesample following at least one reflection from at least one surface ofsaid optically transmissive material.

In embodiments, a cuvette as disclosed herein has a sample chambercomprising an elongated channel. In embodiments, a cuvette as disclosedherein comprises two or more sample chambers for holding sample. Inembodiments, a cuvette may comprise a curved, including U-shaped,channel. In embodiments, a cuvette may comprise a plurality of channels.In embodiments, a sample chamber comprises an inlet port. Inembodiments, a sample chamber comprises a vent effective to allow air orgas to pass in or out (e.g., during filling of the chamber with asample). In embodiments, an inlet port may comprise, or may serve as, avent. In embodiments, a vent may comprise or be covered with a membraneeffective to reduce or prevent evaporation of fluid held within thechannel. In embodiments, an elongated channel of a cuvette may comprisea vent covered with a porous membrane effective to reduce or preventevaporation of fluid held within the channel. In embodiments, anadhesive; a membrane coated on one or two sides with an adhesive layer;ultrasonic welding; or combinations thereof may be used in thefabrication of a cuvette.

In embodiments, a cuvette as disclosed herein may have one or moreoptically non-transmissive surfaces.

In embodiments, trans-illumination may be provided in a cuvette asdisclosed herein, at least in part by internal reflection of lightwithin the cuvette. In embodiments, trans-illumination may be providedin a cuvette as disclosed herein, at least in part by partial internalreflection of light at a surface of the cuvette. In embodiments,trans-illumination may be provided in a cuvette as disclosed herein, atleast in part by total internal reflection of light at a surface of thecuvette.

In embodiments, a cuvette as disclosed herein may have a rectangularhorizontal, cross-sectional shape; in embodiments, a cuvette asdisclosed herein may have a circular horizontal, cross-sectional shape.In embodiments, a cuvette as disclosed herein may have a saw toothvertical cross-sectional shape; in embodiments, a cuvette as disclosedherein may have a step-shaped vertical cross-sectional shape.

Applicants disclose methods herein. For example, Applicants discloseherein a method of identifying a cell in a sample containing a pluralityof cells, comprising: (a) placing said sample in a sample holdercomprising a sample chamber configured to hold the sample, at least aportion of said sample holder comprising an optically transmissivematerial, said optically transmissive material comprising an opticallytransmissive surface and a reflective surface, wherein said opticallytransmissive surface and said reflective surface are configuredeffective that light passing through the optically transmissive surfacesimultaneously provides both epi-illumination and trans-illumination tothe sample in the sample chamber, where epi-illumination comprises lighttraveling from said illumination source to the sample without reflectionat a surface of the optically transmissive material, and wheretrans-illumination comprises light traveling within the opticallytransmissive material and to the sample following at least onereflection from at least one surface of said optically transmissivematerial; (b) illuminating said sample holder effective tosimultaneously provide both epi-illumination and trans-illumination ofthe sample; and (c) identifying a cell in the sample. In embodiments,methods disclosed herein include methods wherein said identifyingcomprises identifying said cell with a detector configured to image atleast a portion of said sample chamber. In embodiments disclosed herein,a sample chamber for use in such methods may comprise an elongatedchannel.

Applicants further disclose herein a method for focusing a microscope,comprising: a) mixing a sample containing an object for microscopicanalysis with a reference particle having a known size, effective togenerate a mixture containing the sample and reference particle; b)positioning the mixture of step a) into a light path of a microscope; c)exposing the mixture of step a) to a light beam configured to visualizethe reference particle; and d) focusing the microscope based on theposition of the reference particle within the mixture or based on thesharpness of an image of the reference particle.

Applicants further disclose herein methods for processing samples,comprising mixing a sample directly with a reagent comprising beads andantibodies, wherein the beads are of a known size and at a knownconcentration, and the antibodies are useful for labeling targets withinthe sample. In embodiments, Applicants disclose methods for processingblood samples, comprising mixing a sample of whole blood with a reagentcomprising beads and antibodies, wherein the beads are of a known sizeand at a known concentration, and the antibodies are useful for labelingblood cells within the sample. Such methods provide improved accuracyand precision of sample analysis, e.g., improved accuracy and precisionof blood cell numbers and characteristics, and reduce the sensitivity ofsample analysis to inaccuracies derived from sample transfer, mixing,and aliquotting. In one non-limiting example, by analyzing the number ofbeads in a sample, one can infer the number of cells if the ratio ofcells-to-beads is known and that ratio is maintained during eachdilution step. It should be understood that every dilution step couldhave variance due to sample dispense and diluent dispense. By startingwith a solution of beads and reagents into which an undiluted sample isadded, the system becomes insensitive to inaccuracies of the dispensesteps so long as the ratio of formed components such as but not limitedbeads and cells does not change.

Applicants disclose herein a method of identifying a cell in a samplecontaining a plurality of cells, comprising: (a) assaying a cell of theplurality of cells for at least one of: (i) the presence of a cellsurface antigen; (ii) the amount of a cell surface antigen; or (iii)cell size; (b) assaying the cell of (a) for at least one of: (i) nuclearsize; or (ii) nuclear shape; and (c) assaying the cell of (a) and (b)for quantitative cell light scatter, wherein the combination ofinformation from steps (a), (b), and (c) is used to identify the cell inthe sample containing a plurality of cells.

In at least one embodiment described herein, a system for imaging asample, the system comprising: a sample vessel containing said sample, astage having a sample vessel receiver with an optically transparentsurface; a light source for illuminating formed components in the samplethrough the stage, wherein the sample vessel has an interface surfaceconfigured to engage the optically transparent surface of the samplevessel receiver whereby the interface surface conforms to the opticallytransparent surface without significant distortion of light passingthrough the interface surface.

It should be understood that embodiments herein may be configured toinclude one or more of the following features. For example, theinterface surface of the sample vessel may be formed from a polymermaterial. Optionally, this may be a transparent material. Optionally,the interface surface of the sample vessel is formed of a materialsofter than a material used to form the optically transparent surface ofthe sample vessel receiver. Optionally, a compression unit is providedfor applying pressure to conform the interface surface to a shapeconfigured to conform with the optically transparent surface of thesample vessel receiver. Optionally, a handling unit may be configured tobe coupled to the sample vessel to facilitate transport of sample vesselon and off the stage, and increase mechanical rigidity of the samplevessel. Optionally, the handling unit may be an optically opaque unitconfigured to be coupled to the sample vessel. Optionally, the handlingunit may be formed with physical features, protrusions, or the like tofacilitate engagement with a robotic manipulator, pipette unit, or othermechanical mover. Optionally, the handling unit may be formed withmagnetic, electromagnetic, or other features to facilitate engagement ordisengagement. Optionally, all imaging of the sample may be done withoutpassing light in a substantially straight line through one surface andout an opposing surface to a detector. Optionally, the light source isnot located on one side of the sample vessel to deliver light to adetector on an opposite side of the sample vessel.

In one non-limiting example, the cuvette may have a plurality ofchannels wherein at least some of the channels have differentcross-sectional widths or other cross-sectional dimensions. Optionally,some cuvettes may also have many different shapes of channels.Optionally, some embodiments may have at least one channel when viewedfrom top-down has a spiral configuration. Optionally, some embodimentmay have a plurality of channels formed as concentric circles,concentric ovals, and/or concentric polygons. Some embodiments may havecuvette channels wherein at least two are of different lengths.

In embodiments, hydrophilic modes of filling or hydrophobic modes offilling may be used with the cuvette. Most microfluidics rely oncapillary action (hydrophilic) for filling channels in a cuvette. Incontrast, at least some embodiments herein may use hydrophobic fillingmodes. In one non-limiting example of hydrophobic mode of filling, aliquid dispensing tip forms a seal with at least one port of thecuvette, and the tip can be used to push the liquid into the cuvettechannel under positive pressure, wherein there is typically a vent atthe end or other portion of the channel in the cuvette to facilitatethis type of liquid filling. By using a hydrophobic surface in all orportions of the channel, one can control how far the liquid goes intothe channel by controlling the pressure. In one non-limiting example ofa cuvette for use in hydrophobic mode of filling, the top layer of thecuvette may be acrylic and the bottom portion of the cuvette is adifferent material. In one embodiment, the bottom portion of the cuvettemay define three sides of the channel (bottom and two sides) while acover layer define the top surface of the channel. Most optically clearmaterials are hydrophobic, so to work with these materials, use of thepressure based filling technique may facilitate filling of these typesof channels.

It should be understood that embodiments in this disclosure may beadapted to have one or more of the features described in thisdisclosure.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a plot of side scatter intensity (x-axis) vs. fluorescenceintensity of a mixture cells including natural killer cells andneutrophils labeled with a fluorescent binder that recognizes CD16.

FIG. 1B shows a bar graph showing the ratio of nuclear area to totalcell area of natural killer cells (“NK”) and neutrophils (“Neu”).

FIG. 1C shows natural killer cells stained with anti-CD16 antibody (leftcolumn) and a nuclear stain (right column).

FIG. 1D shows neutrophils stained with anti-CD16 antibody (left column)and a nuclear stain (right column).

FIG. 2A shows platelets labeled with fluorescently conjugated CD41 andCD61 antibodies (bright dots).

FIG. 2B shows intensity distribution of images of fluorescently labeledplatelets at 10× (left) and 20× (right) magnification.

FIG. 2C shows intensity distribution of an image of a fluorescentlylabeled platelet showing measured intensity (light grey) and curve fitto the measured intensity (dark grey).

FIG. 3 shows: a plot of curve of showing the relationship between thenominal diameter of standard particles in μm (x-axis) and fluorescenceintensity-based size measure in arbitrary units (a.u.; y-axis). Thefigure also shows representative beads at different points along thecurve.

FIG. 4A shows sphered red blood cells imaged by dark field microscopy incuvettes that allow only epi-illumination.

FIG. 4B shows sphered red blood cells imaged by dark field microscopy incuvettes that allow a mixture of epi- and trans-illumination.

FIG. 5A shows putative band neutrophils stained with anti-CD16 antibodyand a nuclear stain.

FIG. 5B shows putative segmented neutrophils stained with anti-CD16antibody and a nuclear stain.

FIG. 6A shows an embodiment of an optical system suitable as part ofdevice or system as disclosed herein, and suitable for use in methodsdisclosed herein, including exemplary optics (e.g., a light-source shownas a ringlight, and an objective), cuvette, and a support structureconfigured to hold and position a cuvette for imaging. In thisembodiment, the cuvette has a rectangular horizontal cross-sectionalshape.

FIG. 6B shows an embodiment of an optical system suitable as part ofdevice or system as disclosed herein, and suitable for use in methodsdisclosed herein, including exemplary optics (e.g., a light-source shownas a ringlight, and an objective), cuvette, and a support structureconfigured to hold and position a cuvette for imaging. In thisembodiment, the cuvette has a circular horizontal cross-sectional shape.

FIG. 7A shows embodiments of elements of an optical system suitable foruse in a device or system as disclosed herein, and suitable for use inmethods disclosed herein.

FIG. 7B shows embodiments of elements of an optical system suitable foruse in a device or system as disclosed herein, and suitable for use inmethods disclosed herein, comprising a further lens and an aperturesuitable for limiting the range of angles of scattered light which reacha detector.

FIG. 8A provides a view of an embodiment of an optical system includinga support structure for holding a cuvette for imaging of a sample, inwhich light from a ringlight illumination system falls directly on thesample (epi-illumination), and light is also reflected from feature ofthe cuvette so as to provide trans-illumination as well. In thisembodiment, the cuvette has a step-shaped vertical cross-sectionalshape.

FIG. 8B provides a view of an embodiment of an optical system includinga support structure for holding a cuvette for imaging of a sample, inwhich light from a ringlight illumination system falls directly on thesample (epi-illumination), and light is also reflected from feature ofthe cuvette so as to provide trans-illumination as well. As shown,incident light may be completely reflected at a surface (total internalreflection, TIR) or only a portion of incident light may be reflected ata surface (partial internal reflection, PIR). In this embodiment, thecuvette has a saw tooth vertical cross-sectional shape.

FIG. 8C shows an embodiment of an optical system suitable as part ofdevice or system as disclosed herein, and suitable for use in methodsdisclosed herein, including exemplary optics (e.g., a light-source shownas a ringlight, and an objective), cuvette, and a support structureconfigured to hold and position a cuvette for imaging. In thisembodiment, the cuvette includes features which affect the path of lightilluminating the cuvette and the sample within the cuvette.

FIG. 8D shows an embodiment of an optical system suitable as part ofdevice or system as disclosed herein, and suitable for use in methodsdisclosed herein, including exemplary optics (e.g., a light-sourcedirecting light from a transverse direction), cuvette, and a supportstructure configured to hold and position a cuvette for imaging. In thisembodiment, the cuvette includes features which affect the path of lightilluminating the cuvette and the sample within the cuvette.

FIG. 8E provides a schematic representation of transport of a cuvettefrom a sample preparation location to a sample observation location nearan optical detector (labeled “D”).

FIG. 8F provides a further, detailed schematic representation of systemincluding a transport mechanism for transporting a cuvette from a samplepreparation location to a sample observation location near an opticaldetector.

FIG. 9A is a dark-field image showing images of representative bloodcells taken from whole blood. FIGS. 9B-9F are also representative imagesof blood cells taken from whole blood, using different imagingtechniques and dyes.

FIG. 9B is an image showing fluorescence from labeled anti-CD14antibodies attached to monocytes.

FIG. 9C is an image showing fluorescence from labeled anti-CD123antibodies attached to basophils.

FIG. 9D is an image showing fluorescence from labeled anti-CD16antibodies attached to neutrophils.

FIG. 9E is an image showing fluorescence from labeled anti-CD45antibodies attached to leukocytes.

FIG. 9F is an image showing leukocyte and platelet cells stained withnuclear stain DRAQ5® (red blood cells, lacking nuclei, are not stainedby DRAQ5®).

FIG. 10 is composite image which shows representative images of bloodcells taken from whole blood, showing a monocyte, a lymphocyte, aneosinophil, and a neutrophil.

FIG. 11A shows identification of monocytes by plotting CD14 labelintensity (FL-17) versus scatter intensity (FL-9). This image, and theother images in FIGS. 11B-11D show plots of fluorescence detected oncells labeled with different markers (labeled antibodies directed atdifferent cell-surface or other markers); such multiple labeling isuseful for identifying cells.

FIG. 11B shows identification of basophils by plotting CD123 intensity(FL-19) versus CD16 intensity (FL-15).

FIG. 11C shows identification of lymphocytes by plotting CD16 intensity(FL-15) versus CD45 intensity (FL-11).

FIG. 11D shows identification of neutrophils and eosinophils by plottingCD16 intensity (FL-15) versus scatter intensity (FL-9).

FIG. 12A plots white blood cell counts obtained by the present methodsversus white blood cell counts obtained by the commercial bloodanalyzer. FIGS. 12A-12F show comparisons of cell counts (measured fromaliquots of the same blood sample) obtained by the present methods, andthose obtained by other methods (using a commercial blood analyzer).

FIG. 12B plots red blood cell counts obtained by the present methodsversus red blood cell counts obtained by the commercial blood analyzer.

FIG. 12C plots platelet counts obtained by the present methods versusplatelet counts obtained by the commercial blood analyzer.

FIG. 12D plots neutrophil counts obtained by the present methods versusneutrophil counts obtained by the commercial blood analyzer.

FIG. 12E plots monocyte counts obtained by the present methods versusmonocyte counts obtained by the commercial blood analyzer.

FIG. 12F plots lymphocyte counts obtained by the present methods versuslymphocyte counts obtained by the commercial blood analyzer.

FIG. 13A shows dark field images of white blood cells (WBCs) obtainedusing microscopy. FIGS. 13A-13E show WBC images obtained usingmicroscopy, for use in performing sequential segmentation analysis todetermine contours for each cell and to thus differentiate the cellimages from the background images.

FIG. 13B is a fluorescence image showing cell labelling by anti-CD45antibodies.

FIG. 13C is a fluorescence image cells labelling by the nuclear stainDRAQ5®.

FIG. 13D is a fluorescence image showing cell labelling by anti-CD16antibodies.

FIG. 13E is a fluorescence image showing cell labelling by anti-CD123antibodies.

FIG. 14A is a dark field image, obtained using microscopy, of whiteblood cells (WBCs). FIGS. 14A-14E show WBC images obtained usingmicroscopy, as in FIGS. 13A-13E, for performing sequential segmentationanalysis to determine external (e.g., cell membrane) and internal (e.g.,nucleus) contours for each cell and to thus identify the cell nucleus aswell as to differentiate the cell regions of interest (cell ROIs) fromthe background regions. The lines within the cell images identify theboundaries of the WBC nucleus for each cell as determined by sequentialsegmentation analysis.

FIG. 14B is a fluorescence image showing cell labelling by anti-CD45antibodies.

FIG. 14C is a fluorescence image cells labelling by the nuclear stainDRAQ5®.

FIG. 14D is a fluorescence image showing cell labelling by anti-CD16antibodies.

FIG. 14E is a fluorescence image showing cell labelling by anti-CD123antibodies.

FIG. 15A is a composite image of the cells shown in FIGS. 13A-13E and14A-14E, with cell contours obtained by watershed segmentation performedonce. FIGS. 15A and 15B show composite images of white blood cells(WBCs) shown in FIGS. 13A-13E and 14A-14E.

FIG. 15B is a the result of sequential segmentation as described hereinapplied to the composite image of the cells shown in FIGS. 13A-13E and14A-14E, showing cell contours obtained by that analysis.

DETAILED DESCRIPTION

Description and disclosure which may aid in understanding the fullextent and advantages of the devices, systems, and methods disclosedherein may be found, for example, in U.S. Pat. No. 7,888,125; U.S. Pat.No. 8,088,593; U.S. Pat. No. 8,158,430; U.S. Pat. No. 8,380,541; PCTApplication No. PCT/US2013/052141, filed Jul. 25, 2013; PCT ApplicationNo. PCT/US2012/057155, filed Sep. 25, 2012; PCT Application No.PCT/US2011/053188, filed Sep. 25, 2011; PCT Application No.PCT/US2011/053189, filed Sep. 25, 2011; U.S. patent application Ser. No.14/098,177, filed Dec. 5, 2013; U.S. patent application Ser. No.13/951,063, filed Jul. 25, 2013; U.S. patent application Ser. No.13/951,449, filed Jul. 25, 2013; U.S. patent application Ser. No.13/769,798, filed Feb. 18, 2013; U.S. patent application Ser. No.13/769,779, filed Feb. 18, 2013; U.S. patent application Ser. No.13/769,818, filed Feb. 18, 2013; U.S. patent application Ser. No.13/769,820, filed Feb. 18, 2013; U.S. patent application Ser. No.13/355,458, filed Jan. 20, 2012; U.S. patent application Ser. No.13/244,947 filed Sep. 26, 2011; U.S. application Ser. No. 13/244,946,filed Sep. 26, 2011; U.S. patent application Ser. No. 13/244,949, filedSep. 26, 2011; U.S. patent application Ser. No. 13/244,950, filed Sep.26, 2011; U.S. patent application Ser. No. 13/244,951, filed Sep. 26,2011; U.S. patent application Ser. No. 13/244,952, filed Sep. 26, 2011;U.S. patent application Ser. No. 13/244,953, filed Sep. 26, 2011; U.S.patent application Ser. No. 13/244,954, filed Sep. 26, 2011; U.S. patentapplication Ser. No. 13/244,956, filed Sep. 26, 2011; U.S. ApplicationSer. No. 61/673,245, filed Sep. 26, 2011; U.S. Patent Application Ser.No. 61/675,811, filed Jul. 25, 2012; U.S. Patent Application Ser. No.61/676,178, filed Jul. 26, 2012; U.S. Patent Application 61/697,797,filed Sep. 6, 2012; U.S. Patent Application 61/766,113, filed Feb. 18,2013; U.S. Patent Application 61/766,116, filed Feb. 18, 2013; U.S.Patent Application 61/766,076, filed Feb. 18, 2013; U.S. PatentApplication 61/786,351, filed Mar. 15, 2013; U.S. Patent ApplicationSer. No. 61/802,194, filed Mar. 15, 2013; U.S. Patent Application Ser.No. 61/837,151, filed Jun. 19, 2013; U.S. Patent Application 61/933,270,filed Jan. 29, 2014; U.S. Patent Application 61/930,419, filed Jan. 22,2014; U.S. patent application Ser. No. 14/161,639, filed Jan. 22, 2014;and U.S. patent application Ser. No. 14/167,264, filed Jan. 29, 2014,the disclosures of which patents and patent applications are all herebyincorporated by reference herein in their entireties.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It may be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a material”may include mixtures of materials, reference to “a compound” may includemultiple compounds, and the like. References cited herein are herebyincorporated by reference in their entirety, except to the extent thatthey conflict with teachings explicitly set forth in this specification.

As used herein, unless explicitly stated otherwise, or unless otherwisemade clear by the context, the meaning of the term “or” includes boththe disjunctive (“or”) and the conjunctive (“and”).

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, if a device optionally contains a feature for a samplecollection unit, this means that the sample collection unit may or maynot be present, and, thus, the description includes both structureswherein a device possesses the sample collection unit and structureswherein sample collection unit is not present.

As used herein, the terms “substantial” means more than a minimal orinsignificant amount; and “substantially” means more than a minimally orinsignificantly. Thus, for example, the phrase “substantiallydifferent”, as used herein, denotes a sufficiently high degree ofdifference between two numeric values such that one of skill in the artwould consider the difference between the two values to be ofstatistical significance within the context of the characteristicmeasured by said values. Thus, the difference between two values thatare substantially different from each other is typically greater thanabout 10%, and may be greater than about 20%, greater than about 30%,greater than about 40%, or greater than about 50% as a function of thereference value or comparator value.

As used herein, “internal reflection” refers to reflection of light,within a material (the first material), at a boundary between the firstmaterial and another material (the second material). For example, afirst material may be a solid such as a glass or plastic, and the secondmaterial may be, e.g., air. The light that is internally reflected istraveling within the first material before it is reflected. Internalreflection may be partial (partial internal reflection: PIR) or total(total internal reflection: TIR). Thus, internal reflection where all ofthe light incident at a surface is reflected back within the firstmaterial is TIR, while internal reflection where not all light incidentat a surface is reflected within a material is PIR. (With PIR, somelight may pass through the boundary, and some light is reflected at thesurface back into the material.) The angle of the incidence is animportant factor in determining the extent of internal reflection; it isthe angle of an incident light ray measured versus a line perpendicularto the boundary surface. Whether or not TIR occurs depends upon theangle of incidence of the light with respect to the surface at theboundary between the first and the second material; the index ofrefraction of the first material; the index of refraction of the secondmaterial; and other factors (e.g., the wavelength of light may affectTIR since the index of refraction typically varies with wavelength). Theangle at which light is totally internally reflected is termed thecritical angle; incident light having an angle of incidence greater thanthe critical angle will be totally internally reflected (will remainwithin the material: TIR). However, with PIR, a portion of incidentlight having an angle of incidence less than the critical angle willalso be internally reflected (the remaining light being refracted andpassing out of the first material into the second material).

As used herein, a “sample” may be but is not limited to a blood sample,or a urine sample, or other biological sample. A sample may be, forexample, a blood sample (e.g., a sample obtained from a finger-stick, orfrom venipuncture, or an arterial blood sample, and may be whole blood,serum, plasma, or other blood sample), a urine sample, a biopsy sample,a tissue slice, stool sample, or other biological sample; a watersample, a soil sample, a food sample, an air sample; or other sample(e.g., nasal swab or nasopharyngeal wash, saliva, urine, tears, gastricfluid, spinal fluid, mucus, sweat, earwax, oil, glandular secretion,cerebral spinal fluid, tissue, semen, cervical fluid, vaginal fluid,synovial fluid, throat swab, breath, hair, finger nails, skin, biopsy,placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavityfluids, sputum, mucus, pus, a microbiota sample, meconium, breast milkor other excretions).

Thus, as used herein, a “sample” includes a portion of a blood, urine,or other biological sample, may be of any suitable size or volume, andis preferably of small size or volume. In some embodiments of thesystems, assays and methods disclosed herein, measurements may be madeusing a small volume blood sample, or no more than a small volumeportion of a blood sample, where a small volume comprises no more thanabout 5 mL; or comprises no more than about 3 mL; or comprises no morethan about 2 mL; or comprises no more than about 1 mL; or comprises nomore than about 500 μL; or comprises no more than about 250 μL; orcomprises no more than about 100 μL; or comprises no more than about 75μL; or comprises no more than about 50 μL; or comprises no more thanabout 35 μL; or comprises no more than about 25 μL; or comprises no morethan about 20 μL; or comprises no more than about 15 μL; or comprises nomore than about 10 μL; or comprises no more than about 8 μL; orcomprises no more than about 6 μL; or comprises no more than about 5 μL;or comprises no more than about 4 μL; or comprises no more than about 3μL; or comprises no more than about 2 μL; or comprises no more thanabout 1 μL; or comprises no more than about 0.8 μL; or comprises no morethan about 0.5 μL; or comprises no more than about 0.3 μL; or comprisesno more than about 0.2 μL; or comprises no more than about 0.1 μL; orcomprises no more than about 0.05 μL; or comprises no more than about0.01 μL.

In embodiments, the volume of sample collected via finger-stick may be,e.g., about 250 μL or less, or about 200 μL or less, or about 150 μL orless, or about 100 μL or less, or about 50 μL or less, or about 25 μL orless, or about 15 μL or less, or about 10 μL or less, or about 10 μL orless, or about 5 μL or less, or about 3 μL or less, or about 1 μL orless.

As used herein, the term “point of service location” may includelocations where a subject may receive a service (e.g. testing,monitoring, treatment, diagnosis, guidance, sample collection, IDverification, medical services, non-medical services, etc.), and mayinclude, without limitation, a subject's home, a subject's business, thelocation of a healthcare provider (e.g., doctor), hospitals, emergencyrooms, operating rooms, clinics, health care professionals' offices,laboratories, retailers [e.g. pharmacies (e.g., retail pharmacy,clinical pharmacy, hospital pharmacy), drugstores, supermarkets,grocers, etc.], transportation vehicles (e.g. car, boat, truck, bus,airplane, motorcycle, ambulance, mobile unit, fire engine/truck,emergency vehicle, law enforcement vehicle, police car, or other vehicleconfigured to transport a subject from one point to another, etc.),traveling medical care units, mobile units, schools, day-care centers,security screening locations, combat locations, health assisted livingresidences, government offices, office buildings, tents, bodily fluidsample acquisition sites (e.g. blood collection centers), sites at ornear an entrance to a location that a subject may wish to access, siteson or near a device that a subject may wish to access (e.g., thelocation of a computer if the subject wishes to access the computer), alocation where a sample processing device receives a sample, or anyother point of service location described elsewhere herein.

The term “cells,” as used in the context of biological samples,encompasses samples that are generally of similar sizes to individualcells, including but not limited to vesicles (such as liposomes), cells,virions, and substances bound to small particles such as beads,nanoparticles, or microspheres.

As used herein, the term “binds” refers to a reaction, or interaction,between two materials which lead to the close combination of the two;e.g., a reaction between a ligand and a receptor, in which the ligandbecomes tightly linked to the receptor, provides an example of binding.The combination of an antibody with its target antigen, and of a carrierprotein with its cargo, such as intrinsic factor with vitamin B12, arefurther examples of reactions in which one material binds to another.

The term “binder” as used herein refers generally to any compound ormacromolecule, such as an antibody, which tightly or specifically bindsto a target. Binders include, but are not limited to, antibodies(whether monoclonal or polyclonal, antibody fragments, immunoadhesins,and other such antibody variants and mimics), natural binding proteins(e.g., intrinsic factor protein which is specific for vitamin B12),ligands which bind their target receptors, substrates which bind toparticular enzymes, binding pairs such as avidin and biotin, smallmolecules which tightly and specifically bind to target molecules, andthe like. Bacteria, viruses, synthetic scaffolds, and other objects andmaterials that bind or adhere to specific targets may be used asbinders. A binder may be, or may include, or may be linked to, a markersuch as a dye, or fluorophore, or other detectable moiety.

As used herein, a “marker” is a detectable material whose presence makesa target visible or otherwise detectable, or whose presence in aposition or location is indicative of the presence of a target in thatposition or location. A marker may be used to label a cell, structure,particle, or other target, and may be useful to detect, determine thepresence of, locate, identify, quantify, or otherwise measure a targetin, or property of, a sample. Markers may include, without limitation,stains, dyes, ligands, antibodies, particles, and other materials thatmay bind or localize to specific targets or locations; bacteria, virusesor cells that may grow in or localize to specific targets or locationsmay also be used as markers. Detectable attributes or properties ofcells or other targets may be used as markers.

As used herein, the terms “stain” and “dye” may be interchangeable, andrefer to elements, compounds, and macromolecules which render objects orcomponents of a sample more detectable than in the absence of treatmentwith the stain or dye. For example, treatment of a blood sample with aDNA dye such as propidium iodide renders the nuclei of nucleated cellsmore visible, and makes detection and quantification of such cellseasier than otherwise, even in the presence of non-nucleated cells(e.g., red blood cells).

As used herein, the term “surfactant” refers to a compound that iseffective to reduce the surface tension of a liquid, such as water. Asurfactant is typically an amphiphilic compound, possessing bothhydrophilic and hydrophobic properties, and may be effective to aid inthe solubilization of other compounds. A surfactant may be, e.g., ahydrophilic surfactant, a lipophilic surfactant, or other compound, ormixtures thereof. Some surfactants comprise salts of long-chainaliphatic bases or acids, or hydrophilic moieties such as sugars.Surfactants include anionic, cationic, zwitterionic, and non-ioniccompounds (where the term “non-ionic” refers to a molecule that does notionize in solution, i.e., is “ionically” inert). Exemplary commerciallyavailable amphiphilic compounds include Tergitol™ nonionic surfactants;Dowfax™ anionic surfactants; polyethylene glycols and derivativesthereof, including Triton™ surfactants; polysorbates(polyethylenesorbitans) such as the TWEEN® compounds, and poloxamers(e.g., ethylene oxide/propylene oxide block copolymers) such asPluronics® compounds; stearates and derivatives thereof; laurates andderivatives thereof; oleates and derivatives thereof; phospholipids andderivatives thereof; lysophospholipids and derivatives thereof; sterolsand derivatives thereof; and combinations thereof.

As used herein, a “detector” may be any device, instrument, or systemwhich provides information derived from a signal, image, or otherinformation related to a target, such as a sample. Detectable signalsand information may include, for example, optical, electrical,mechanical, chemical, physical, or other signals. A detector may be, forexample, an optical detector, or an electrical detector, or a chemicaldetector, or an electrochemical detector, or an acoustic detector, or atemperature detector, or a mechanical detector, or other detector.

As used herein, an “optical detector” detects electromagnetic radiation(e.g., light). An optical detector may detect an image or be used withan image, or may detect light intensity irrespective of an image, orboth. An optical detector may detect, or measure, light intensity. Someoptical detectors may be sensitive to, or restricted to, detecting ormeasuring a particular wavelength or range of wavelengths. For example,optical detectors may include, for example, photodiode detectors,photomultipliers, charge-coupled devices, laser diodes,spectrophotometers, cameras, microscopes, or other devices which measurelight intensity (of a single wavelength, of multiple wavelengths, or ofa range, or ranges, of wavelengths of light), form an image, or both.

The term “ploidy” as used herein refers to the amount of DNA in a cell,and to assays and measurements of the DNA content of cells in a sample.Ploidy measurements provide a measure of whether or not a cell, or apopulation of cells, has a normal or an abnormal amount of DNA, or,since DNA is duplicated during cell division and proliferation, ifabnormal numbers of cells in a population are proliferating. Ploidymeasurements may be made by imaging techniques following staining ofnucleated cells in a sample with a DNA-specific dye.

Quantitative Microscopy

In some embodiments, methods, systems, and devices are provided hereinfor quantitative microscopy. Quantitative microscopy may involve one ormore of quantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy methods to measure one or more cellular attributes.Any of these methods may provide morphometric information regardingcells. Such information may be measured quantitatively. In someembodiments, for quantitative microscopy, a sample is analyzed by two ormore of quantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy. Quantitative microscopy may include use of imageanalysis techniques or statistical learning and classification methodsto process images obtained by microscopy.

Multiple different cellular attributes may be measured duringquantitative microscopy. Cellular attributes that may be measuredinclude, without limitation:

Physical attributes: e.g. cell size, volume, conductivity, low and highangle scatter, and density. Other physical attributes that may bemeasured or quantified include, without limitation, circularity of acell or particle; aspect ratio of a cell or particle; perimeter of acell or particle; convexity of a cell or particle; granularity of a cellor particle; intensity of an image of a cell or particle; height (e.g.,size through several focal planes) of a cell or particle; flatness of acell or particle; and other physical attributes.

Morphological attributes: e.g. cell shape, area, size, and lobularity;nucleus shape area, size, and lobularity; mitochondria shape, area,size, and lobularity; and ratio of nuclear volume to cell volume.

Intracellular attributes: e.g. nucleus centroid/cell centroid distance(i.e. distance between the center of the nucleus and the center of thecell), nucleus lobe centroid distance (i.e. distance between the centerof different lobes of the nucleus), distribution of proteins within thecells (e.g. actin, tubulin, etc.), distribution of organelles within thecells (e.g. lysosomes, mitochondria, etc.), colocalization of proteinswith other proteins and organelles, and other attrtibutes.

Biochemical attributes: e.g. expression level of cellular proteins, cellsurface proteins, cytoplasmic proteins, nuclear proteins, cellularnucleic acids, cell surface nucleic acids, cytoplasmic nucleic acids,nuclear nucleic acids, cellular carbohydrates, cell surfacecarbohydrates, cytoplasmic carbohydrates, and nuclear carbohydrates.

In some embodiments, methods, systems, and devices are provided hereinfor the quantitative measurement of two, three, four, five or moreattributes of cells in a sample, wherein the attributes are selectedfrom physical attributes, morphological attributes, intracellularattributes, and biochemical attributes. In some embodiments, methods,systems, and devices are provided herein for the quantitativemeasurement of two, three, four, five or more attributes of cells in asample, wherein the attributes are selected from: cell size, cellvolume, cell conductivity, cell low angle light scatter, cell high anglelight scatter, cell density, cell shape, cell area, cell lobularity,nucleus shape, nucleus area, nucleus size, nucleus lobularity,mitochondria shape, mitochondria area, mitochondria size, mitochondrialobularity, ratio of nuclear volume to cell volume, nucleuscentroid/cell centroid distance, nucleus lobe centroid distance,distribution of proteins with the cells (e.g. actin, tubulin, etc.),distribution of organelles within the cells (e.g. lysosomes,mitochondria, etc.), expression level of a cellular protein, expressionlevel of a cell surface protein, expression level of a cytoplasmicprotein, expression level of a nuclear protein, expression level of acellular nucleic acid, expression level of a cell surface nucleic acid,expression level of a cytoplasmic nucleic acid, expression level of anuclear nucleic acid, expression level of a cellular carbohydrate,expression level of a cell surface carbohydrate, expression level of acytoplasmic carbohydrate, and expression level of a nuclearcarbohydrate.

In some embodiments, methods are provided for the quantitativemeasurement of two, three, four, five, or more attributes of cells in abiological sample by microscopy, wherein the method may include one ormore of the following steps or elements. The attributes of the cellsquantitatively measured may be selected from the attributes listed inthe immediately above paragraph. The biological sample may bepre-treated prior to microscopy. Pre-treatment may include any procedureto aid in the analysis of the sample by microscopy, including: treatmentof the sample to enrich for cells of interest for microscopy, treatmentof the sample to reduce components in the sample which may interferewith microscopy, addition of material to the sample to facilitateanalysis of the sample by microscopy (e.g. diluents, blocking moleculesto reduce non-specific binding of dyes to cells, etc.). Optionally,prior to microscopy, a sample may be contacted with one or more bindersthat specifically bind to a cellular component. Binders may be directlylinked to a dye or other particle for the visualization of the binder. Asample may also be contacted with a secondary binder, which binds to thebinder which binds to the cellular component. A secondary binder may bedirectly linked to a dye or other particle for the visualization of thebinder. Prior to microscopy, a sample may be assayed in aspectrophotometer. For microscopy, a biological sample containing orsuspected of containing an object for microscopic analysis may beintroduced into a sample holder, such as a slide or a cuvette. Thesample holder containing a sample may be introduced into a deviceconfigured to perform quantitative microscopy on the sample. Themicroscope may be coupled with an image sensor to capture imagesgenerated through the microscope objective. In the device, multipleimages of the sample may be acquired by microscopy. Any one or more ofquantitative fluorescence microscopy, quantitative dark fieldmicroscopy, quantitative bright field microscopy, and quantitative phasecontrast microscopy may be used to obtain images of the sample.Optionally, images of the entire sample in the sample holder may beacquired by microscopy. Multiple fields of view of the microscope may berequired to capture images of the entire sample in the sample holder.The sample holder may move relative to the microscope or the microscopemay move relative to the sample holder in order to generate differentfield of views in order to examine different portions of the sample inthe sample holder. Multiple images of the same field of view of thesample in the sample holder may be acquired. Optionally, multiplefilters may be used with the same type of microscopy and the same fieldof view of the sample, in order to acquire different images of the samesample which contain different information relating to the sample.Filters that may be used include, without limitation bandpass and longpass filters. Filters may permit the passage of certain wavelengths oflight, and block the passage of others. Optionally, multiple types ofmicroscopy (e.g. fluorescence, dark field, bright field, etc.) may beused to acquire images of the same field of view of the sample, in orderto acquire different images of the same sample which contain differentinformation relating to the sample. Optionally, video may be used tocollect microscopy images. Optionally, microscopy images may becollected in 3-D. For microscopy performed as described herein, thedevice or system may be configured to link information relating to acell in one image of the sample to the same cell in a different image ofthe sample. Based on different images of the same sample or same cells,multiple attributes of cells in the sample may be determined. In someaspects, the combination of multiple attributes/multiple pieces ofinformation about cells in a sample may be used to reach a clinicaldecision or to draw a conclusion about the cells that would not bepossible based on information from only a single attribute of the cells.

In some embodiments, devices and systems are provided for thequantitative measurement of two, three, four, five, or more attributesof cells in a biological sample by microscopy. In some embodiments, thedevice or system contains both a microscope or cytometer and aspectrophotometer. The device or system may further contain a fluidhandling apparatus, which is configured to move sample between aspectrophotometer and a microscope or cytometer. In some embodiments,devices and systems for performing the methods disclosed herein areconfigured as described in U.S. patent application Ser. No. 13/244,947and U.S. patent application Ser. No. 13/769,779, which are each herebyincorporated by reference in their entireties. Although the foregoinghas been described in the context of a cell, it should also beunderstood that some or all of the foregoing may also be applied tocrystals, particles, filaments, or other cell-sized objects that may befound in a sample.

Dynamic Dilution

In some embodiments, methods, systems, and devices are provided hereinfor dynamic dilution of cell-containing samples.

By way of non-limiting example, a method for dynamic dilution of asample may include one or more of the following steps or elements suchthat a desired number or concentration of cells or objects in the sampleis determined and this information is used as a factor in adjustingdownstream sample processing. In this non-limiting example, one or morestains or dyes may be added to a biological sample containing cells. Themixture of stain and sample may be incubated. The cells in the mixtureof stain and sample may be washed to remove excess (unbound) stain. Thestained, washed cells may be prepared in a desired volume for furtheranalysis. The stained, washed cells may be analyzed to determine theapproximate number or concentration of cells in the sample or a portionthereof. Based on the number or concentration of stained cells in thesample or portion thereof, a volume of sample may be obtained forfurther analysis, such that a desired number or concentration of cellsfor further analysis is obtained. In some embodiments, samples may bediluted as described in U.S. patent application Ser. No. 13/355,458,which is hereby incorporated by reference in its entirety.

In one embodiment as described herein, it is desirable to provideanother detection technique such as but not limited tofluorescence-based method for enumerating cells, to estimate cellconcentration in place of using a cell counter. This estimate isdescribed because, for accurate and reproducible staining of patientsamples, it is often desirable that stains (DNAdyes/antibodies/binders/etc.) are optimally titered for a specificnumber/concentration of cells. For example, a known concentration ofstain will be applied to a specific number of cells (e.g. 0.2 microgramsof stain per one thousand white blood cells (WBCs)). After an incubationperiod, the sample will be washed to remove excess (unbound) dye,prepared at the appropriate cell density, and imaged.

In this non-limiting example, to make an estimate of cell concentrationfor a targeted cell type, a sample is non-destructively measured with adifferent modality from that used for cytometry, such as but not limitedto a spectrophotometer, in order to inform sample processing for thecytometric assay. The method may comprise selecting another markerunique to the cell population of interest. In one non-limiting example,for B-cells, one may choose CD20. The process comprises labeling thesample with anti-CD20 binders conjugated to a different coloredfluorophore than CD5. One then measures the fluorescent signal of thissample non-destructively and rapidly using a device such as but notlimited to a fluorescence spectrophotometer. Using calibration, it ispossible to predict the concentration of B-cells with limited accuracyto provide the estimate. In one non-limiting example, the calibrationmay correlate signal strength with the number of cells for that type ofsignal. The creation of these calibration curves can be used to estimatethe number of cells or object. Other techniques for estimating number ofcells based on an overall signal strength such as but not limited tooptical, electrical, acoustical, or the like are not excluded. Based onthe approximate concentration of B-cells, the system can estimate theappropriate amount and concentration of anti-CD5 binder so thatproportional relationship between CD5 expression and CD5 fluorescence ismaintained. In this manner, the stain and staining procedure can beoptimized/normalized for a particular cell number.

To maximize the use of patient samples (which may be low volume samples,such as, e.g., blood obtained from a finger-stick, having a volume equalto or less than about 120 μL), it is desirable to develop methodswhereby the number of WBCs contained within a given volume of blood canbe enumerated (e.g., the concentration WBCs/μL determined). This allowsthe number of WBCs to be determined, or at least estimated, prior toadding stains. Once determined, a desired number of cells can bealiquotted for incubation with a known concentration of stain(s),yielding optimal resolution of cell subpopulations.

In an application where measurement of ploidy of cells is desired, cellsin a sample may be stained with a DNA dye, and then the intensity ofstaining may be quantified (where “the intensity of staining” means theintensity of an optical signal due to the dye). The intensity of the dyesignal due to such staining depends upon the ratio of DNA/dye (that is,of the amount of DNA stained by the dye to the amount of dye added). Ifa preset amount of dye is added to every sample, regardless of thecharacteristics of the sample, then samples with very high cellconcentration will each be less bright as compared to samples with lowcell concentration. This situation would confound the quantification ofthe amount of DNA in each cell. As disclosed herein, obtaining anestimate of the number of nucleated cells in a sample prior to addingthe dye allows one to adjust the amount of dye so that quantification ofthe DNA, and of the amount of DNA per cell in the sample, may beperformed. Thus, for example, a sample, or an aliquot of a sample, maybe treated with a stain or dye directed at a cell-surface markerindicative of the cell or cells to be quantified, and that surfacemarker used to non-destructively estimate the concentration of cells inthe sample. This estimated concentration may then be used to calculatethe amount of dye that needs to be added to the sample so as to alwaysmaintain a consistent DNA:Dye ratio (mole to mole) for subsequentmeasurements.

In a first example of a fluorescence-based method for enumerating cells,a method may comprise determining the ploidy of cells (e.g., enumeratingcells via fluorophore-conjugated antibody staining) In this non-limitingexample, it is desired to enumerate the WBCs in a blood sample so that aspecific number of WBCs can be stained with a predeterminedconcentration of DNA dye (e.g., 4′-6-diamidino-2-phenylindole (DAPI), or1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione(DRAQ5®), or propidium iodide, or other DNA-staining dye). The method ofthis example comprises counting WBCs using a fluorophore-conjugatedantibody and a spectrophotometer. It should be understood that thisapproach may be helpful when staining cells with a DNA dye anddetermining ploidy, where the ratio of cell number to DNA dyeconcentration (cell#: [DNA dye]) is desirable for generating comparableand consistent data. Given that the number of cells per microliter ofblood vary within a healthy population, it is typically desirable todetermine the number of WBCs per microliter before attempting to stainfor ploidy.

In an embodiment, the procedure comprises using cells that are firststained with a fluorophore-conjugated antibody (where the antibody ispreferably directed to a ubiquitously expressed antigen, such as CD45,or to a subpopulation specific antigen, such as CD3 for T cells), orfluorescent dye which labels all cells (e.g., a membrane or cytoplasmicstain such as eosin, or a lectin or other stain or dye) where thewavelength of the fluorescence from the fluorophore is spectrallydistinct (and preferably distant) from the emission wavelength of theDNA dye. After an incubation period, the sample is washed to removeexcess (unbound) antibody, prepared in the appropriate volume, andanalyzed via a spectrophotometer. The resulting data allows the numbersof WBCs in a blood sample to be determined, so that a specific volume ofblood can be aliquotted (yielding a particular/desired number of WBCs)and stained with a DNA dye. The resulting data is useful to calculateand to adapt the amount of DNA dye to be used in staining a sample,according to the number of WBCs determined using thefluorophore-conjugated antibody as described.

A further embodiment comprises determining the number of cells (via DNAstaining) prior to surface staining of the cells. Additional details mayalso be found in the cell enumeration section herein below. It issometimes desirable to enumerate the WBCs in a blood sample so that aspecific number of WBCs can be stained with optimal concentrations ofantibodies. In one embodiment, the method comprises counting WBCs usinga DNA dye and a spectrophotometer, e.g., as discussed above.

Alternatively, if the number of cells per microliter was determinedprior to staining, then a known number of cells could be aliquotted andstained for each sample, regardless of (i) variation within a healthypopulation and (ii) disease state. To determine the number of cells permicroliter of blood, it may be possible to use DNA dyes such as DAPI,DRAQ5®, or propidium iodide. Optionally, unbound dye may be washed away.A spectrophotometer can be used to determine the number of nucleated(e.g., DRAQ5® positive) cells per microliter of blood.

The number and concentration of white blood cells (WBCs) in equal-sizedaliquots of blood may vary from subject to subject. However, foradequate analysis of WBCs in a blood sample, sufficient amounts ofreagents (such as antibodies targeting particular WBC-specific antigens)may be added, and the amount that is sufficient depends upon the numberand concentration of WBCs in a blood sample. A procedure termed “dynamicdilution” may be used to ensure that the sufficient antibody reagent isadded to a sample. In one non-limiting example, the procedure treatsblood cells in order to obtain a provisional cell count used to gaugethe proper amount of reagent (e.g., an antibody cocktail for stainingwhite blood cells (WBCs)) to be used with the sample in order to providecomplete staining of the cells. In the procedure, the cells are stainedwith a DNA dye (e.g., DAPI, DRAQ5, or propidium iodide) that isspectrally distinct/distant from the emission of thefluorophore-conjugated antibodies that will be used in subsequent stepsor assays. Optionally, the sample may be washed to remove excess(unbound) DNA dye after an incubation period. After an incubationperiod, the sample may be prepared in the appropriate volume, and imagedor measured using a spectrophotometer. The resulting data allows thenumber of WBCs in the known volume of sample to beenumerated/determined, so that a specific volume of blood can bealiquotted (yielding a particular/desired number of WBCs) and stainedwith the proper amount of antibodies (i.e., based on the estimatednumber of WBCs determined using the DNA dye, the amount of antibodiesmay be determined that are required in order to provide the desiredsaturation of antibody staining) Thus, the estimate provided by the DNAstaining allows calculation and addition of the proper amount ofantibody dye required for the number of WBCs in the sample aliquot.

Dynamic Dilution Protocol:

In one embodiment, a dynamic dilution protocol involves taking analiquot of a blood sample containing white blood cells (WBCs) (e.g.,whole blood, or a blood portion containing WBCs) in order to estimatethe amount of reagent containing antibodies targeting the WBCs that isneeded for analysis of the sample.

In this non-limiting example, a known volume of a blood sample is taken.A known amount of nuclear dye (e.g., a DNA-staining dye such aspropidium iodide, DAPI, or Draq5®) is added to this known volume sample.The mixture is then incubated for a period of 2 to 10 minutes at atemperature between 25° C. to 40° C.

Next a red blood cell (RBC) lysis buffer is added. In this non-limitingexample, the mixture is then incubated for a period of 2 to 10 minutesat a temperature between 25° C. to 40° C. (lower temperatures may alsobe used). A suitable lysis buffer may be, for example, a hypotonicsaline solution; a hypotonic sucrose solution; an isotonic ammoniumchloride solution; an isotonic solution including a gentle surfactantsuch as saponin or other buffer in which RBCs will lyse. Othersurfactants disclosed herein may be used; for example, surfactants whichmay be suitable for use in a lysis buffer include, without limitation,polysorbates (e.g., TWEEN™), polyethylene glycols (e.g., Triton™surfactants), poloxamers (e.g., PLURONICS™), detergents, and otheramphiphilic compounds. In embodiments, such lysis buffers will include afixative (such as, e.g., formaldehyde, paraformaldehyde, glutaraldehyde,or other fixative) to aid in stabilizing WBCs. A surfactant such assaponin causes a large number of holes to be formed in the membranes ofcells. Red blood cells, due to their unique membrane properties, areparticularly susceptible to this hole formation and lyse completely,their contents leaking out into the liquid around. The presence of afixative prevents unintentional lysis of the white blood cells.Platelets also remain unlysed. The purpose of this step is to removeintact red blood cells from the mixture as they outnumber white bloodcells by about 1000:1. Platelets do not interfere with imaging and henceare not a consideration in this process. In embodiments, a lysis buffermay also contain non-fluorescent beads at a known concentration; thesebeads may serve as size or concentration markers. The lysis of the RBCs,along with the subsequent steps of this protocol, substantially removesany RBC interference to imaging or to optical measurements of the WBCs.Such optimization of the ratio of lytic agent to fixative (e.g., saponinto paraformaldehyde) provides effective lysis of RBCs with a minimalvolume of lysis buffer and with minimal adverse effects on WBCs (orplatelets) in a sample. By increasing both lytic agent and fixativeconcentration (e.g., saponin and paraformaldehyde concentrations,respectively) Applicants have been able to reduce the concentration oflysis buffer to sample volume from approximately 20:1 to about 4:1(lysis buffer volume:sample volume). Further increases in lytic agentconcentration risks excessive increasing of WBC lysis as well as thedesired lysis of RBCs.

Next the treated sample is separated, where the separation may beperformed by any suitable method, such as but not limited to spinningthe treated sample in a centrifuge at 1200×g for 3 minutes.

Following separation (e.g., centrifugation), the supernatant is removed;the remaining pellet is then resuspended. In embodiments, the pellet isresuspended in some or all of the supernatant. A known volume ofsolution containing the resuspended pellet results from this step.

If desired, a further separation step, and a further resuspension step,may be performed. These steps provide a concentrated sample with cellsthat are approximately 10-fold concentrated (ignoring any possible celllosses at each step).

The amount of DNA-staining dye in the resuspended, concentrated sampleis then measured. For example, the fluorescence from a fluorescentDNA-staining dye such as DRAQ5® may be measured in a spectrophotometer.In embodiments, the sample may be illuminated by light at a wavelengthof 632 nm (the excitation wavelength of DRAQ5®), the light emitted bythe cell suspension may be filtered by a 650 nm long pass filter, andthen the emitted light may be measured in a spectrophotometer. Thisemission measurement is then correlated with a previously generatedcalibration curve to estimate a rough concentration of white blood cellsin the cell suspension. Typically, cell concentrations have ranged fromabout 1000 cells per microliter to about 100,000 cells per microliter.The estimate of WBC number obtained in this way may be used to calculatean appropriate dilution factor to ensure that the concentration of cellsin the sample, when used in subsequent quantitative measurements, isconstrained to within a range (e.g., a two-fold or other range) around apre-defined target concentration. The sample is then diluted per thecalculated dilution factor to provide a sample with a WBC concentrationwithin the desired concentration range.

The purpose of this “dynamic dilution” step is to ensure that WBCs arenot present at too high or too low a concentration in the sample. If thecell concentration is too high, the accuracy of image processingalgorithms is compromised, and if the cell concentration is too low, aninsufficient number of cells are sampled. Dilution of a concentratedsample as disclosed herein provides WBC concentrations within a desiredrange and ensures that signals from the sample during analysis will fallwithin an optimum range for detection and analysis.

In addition, estimation of the number of WBCs in this way allows thecalculation (within a small range) of the amounts of reagents requiredfor further assays and method steps applied to the sample, since thenumbers of WBCs in a sample may vary, yet the amount of reagent requiredfor the various assays may depend upon the number of WBCs in the sampleto be assayed. For example, the reagents to be added after estimation ofWBC number by the dynamic dilution protocol include antibodies thattarget specific antigens found on different types of WBCs, or, if theseantigens are found on multiple types of WBCS, which are present indiffering amounts on different types of WBCs. In the absence of such anestimate of the number of WBCs in a sample, predetermined amounts ofdyes and other reagents must be used in subsequent assays of the sample,leading to incorrect amounts of reagents and inaccurate or incompleteassay results. Thus, this Dynamic Dilution Protocol serves as animportant and useful initial step in the full assessment of a bloodsample from a patient, and allows for more precise and accuratemeasurements to be made than would be possible otherwise.

Dynamic Staining

In some embodiments, methods, systems, and devices are provided hereinfor dynamic staining of cell-containing samples.

Measurement of a Component of Interest in Cells of a Cellular Population

In one embodiment, a method for dynamically staining a cell samplerelates to a method for the measurement of a component of interest incells of a cellular population in a sample.

As used herein, a “component of interest” refers to any type of moleculethat may be present in a cell. “Components of interest” includeproteins, carbohydrates, and nucleic acids. Typically, a “component ofinterest” is a specific species of molecule, such as a particularantigen. Non-limiting examples of “components of interest” of a cellinclude: CD5 protein, CD3 protein, etc.

As used herein, a “cellular population” refers to any grouping of cells,based on one or more common characteristics. A “cellular population” mayhave any degree of breadth, and may include a large number of cells oronly a small number of cells. Non-limiting examples of “cellularpopulations” include: red blood cells (RBCs), white blood cells,B-cells, CD34+B-cells, etc.

In some circumstances, it may be desirable to quantitatively measure acomponent of interest in cells of a certain cellular population in asample from a subject. For example, it may be desirable to measure theextent of CD5 (the “component of interest”) expression in B-cells (the“cellular population”) in a sample of cells from a subject havingchronic lymphocytic leukemia. Detection or measurement of the level of acomponent of interest may involve use of a binder molecule that hasaffinity for the specific component of interest, such an antibody orsingle chain variable fragment (“scFv”). In order to accurately measurethe level of a specific component of interest in cells in a methodinvolving the use of a binder molecule, it may be advantageous to exposethe cells to the binder molecule at a specific ratio or range of ratiosof binder molecule to target component of interest. For example, it maybe desirable to provide an amount of binder to a collection of cellssuch that there is a linear relationship between the amount of componentof interest in the cells and the amount of binder which binds to thecomponent of interest in the cells. For example, it may be undesirableto have too little binder (such that there is not enough binder to bindto all of the components of interest in the cells) or to have too muchbinder (such that the binder binds non-specifically to the cells).

Using traditional methods, it may be difficult to provide an appropriatelevel of binder to a sample in order to accurately measure the amount ofcomponent of interest in a cellular population in the sample, due to thefact that the size of the cellular population or component of interestin the sample may vary significantly between different samples. Incontrast, provided herein are methods, devices, and systems fordynamically staining cell samples to accommodate samples containing awide range of cellular populations and components of interest.

In one embodiment, a method for the measurement of a component ofinterest in cells of a cellular population in a sample is provided. Themethod is not limited to but may include one or more of the followingsteps.

First, a quantitative or semi-quantitative measurement of a markerpresent in cells of the cellular population may be obtained. The markermay be any marker which is present in the cellular population ofinterest, and it may be a marker exclusively present in the cellularpopulation of interest (i.e. not present in any other cell types in thesample). Measurement of the marker may be by any method, provided themethod does not destroy the sample, and may use any system or device. Abinder which recognizes the marker may be mixed with the sample. Thebinder may have a molecule attached to facilitate detection of thebinder (e.g. a fluorescent marker). In an example, the marker may bedetected or measured by fluorescence spectrophotometry. In embodimentsin which the binder has a fluorescent label and the marker is measuredby fluorescence spectrophotometry, fluorescence spectrophotometry may beused to measure a bulk fluorescence from the sample or a portionthereof, rather than to measure fluorescence from individual cells.

Second, based on the quantitative or semi-quantitative measurement ofthe marker present in cells of the cellular population, an approximateamount or concentration of cells of the cellular population present inthe sample may be determined. The approximate number or concentration ofcells in the cellular population present in the sample may bedetermined, for example, through the use of a calibration curve.Calibration curves may be prepared or may be available for differentmarkers/binder combinations. Calibration curves may be developed, forexample, by measuring the signal from known numbers of cells having acertain marker and bound with a certain binder. In some embodiments, theapproximate amount or concentration of cells of the cellular populationpresent in the sample may be determined with the aid of a computer. Insome aspects, the approximate number or concentration of cells in thecellular population present in the sample may be determined, with such adetermination being no more than about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 150, 200, 250, 300, 400, or 500% off the true concentration.

Third, based on the determined amount or concentration of cells in thecellular population present in the sample, an amount of a reagent to addto the sample may be selected, wherein the reagent binds specifically tothe component of interest in cells of the cellular population. Thereagent may be or include any molecule that binds specifically to thecomponent of interest. For example, the reagent may be a binder, such asan antibody. The reagent may be configured such that it may be readilydetected (e.g. by fluorescence or luminescence) or such that under atleast some circumstances, it produces a detectable signal. In someembodiments, the reagent may be attached to a molecule to facilitatedetection of the reagent. The amount of reagent added to the sample maybe any amount. In some embodiments, an amount of reagent may be added tothe sample such that there is an approximately linear relationshipbetween the level of the component of interest in individual cells ofthe cellular population and the signal produced by the reagents bound tothe components of interest in individual cells of the cellularpopulation.

Fourth, after the amount of a reagent to add to the sample is selected,the selected amount of reagent may be added to the sample.

Fifth, cells in the sample may be assayed for reagent bound to thecomponent of interest.

Sixth, based on the amount of reagent bound to the component ofinterest, the amount of the component of interest in cells of thecellular population of the sample may be determined.

In some embodiments, the fifth and sixth steps may be performed togethersuch that the measurement of the amount of reagent bound to thecomponent of interest is sufficient to identify the amount of thecomponent of interest in cells of the cellular population of the sample.

In other embodiments, provided herein are systems and devices for thedynamic staining of samples. The systems and devices may contain,without limitation, a spectrophotometer and a fluorescence microscope.In an embodiment, a system or method for dynamic staining of samples maybe configured as described in U.S. patent application Ser. No.13/244,947 or Ser. No. 13/355,458, which are hereby incorporated byreference in their entirety. In an embodiment, the systems and devicesmay be automated to determine an amount of a reagent to add to a sampleto determine the amount of a component of interest in cells of acellular population in a sample, based on a measurement of an amount ofa marker present in cells of the cellular population. In anotherembodiment, the systems and devices may be automated to determine anamount of a reagent to add to a sample to determine the amount of afirst component in cells of a cellular population in a sample, based ona measurement of an amount of a second component in the cells of thecellular population in a sample.

Context-Based Autofocus

In some embodiments, methods, systems, and devices are provided hereinfor context-based microscopy autofocus.

The size (e.g., length, height, or other measure) of many clinicallyrelevant objects in biological samples spans a wide range. For example,bacteria are commonly about 1 μm in length, erythrocytes are commonlyabout 6-8 μm in length, leukocytes are commonly about μm 10-12 inlength, epithelial cells may be about 100 μm in length, and cast andcrystals may be about 200-300 μm in length. In addition, there are manyamorphous elements such as urinary mucus which exist as strands orfilaments which may range from about 10-400 μm in length.

A challenge in microscopy is to acquire precise images of fields of viewthat contain an unknown and arbitrary composition of objects of varioussizes, such as those described above. Since the depth of focus of manymicroscopy objectives is limited (typically about 1-10 μm), for a givenfield of view containing elements of various sizes, multiple focalplanes for the given field of view may need to be acquired in order toobtain accurate sharp images of the various elements within the field ofview. A problem with many traditional autofocus methods is that they aredesigned to focus on the dominant feature in a field of view, so thatthe sharpness of that feature can be maximized. Such methods may beineffective for capturing elements of various sizes in a sample.

In one embodiment, a method is provided for context-based microscopyautofocus, which includes mixing a reference particle of a known sizewith a sample for microscopy. In embodiments, more than one referenceparticle is added to the sample; preferably all, or substantially all,of such reference particles are of the same known size. In embodiments,the number of reference particles added to a particular volume of sampleis known. The reference particles may be detected during microscopy, andused to achieve focusing. By use of the reference particles to achievefocusing, focal planes may be selected independent from the overallimage composition. In one aspect, the method may be useful to achievefocusing on a sample having an unknown composition of elements. Inanother aspect, the method may support the generation of precise planesof focus, independent of the precision of the microscope ormicroscopy-related hardware. For example, when a plane of focus isselected based on feedback from the sharpness of the reference particleswithin a field of view, precise focusing on various elements within asample may be achieved, regardless of the level of accuracy or precisionof the focusing hardware [e.g. the microscope objective actuation, theshape of a sample holder (e.g. a cuvette or slide), or thenon-uniformity of a sample holder].

In an embodiment, a reference particle may contain or be labeled with amolecule to facilitate detection of the particle during microscopy. Inone example, a reference particle may be labeled with or contain afluorescent molecule. The fluorescent molecule may absorb light at afirst wavelength of light, and, in response to the absorbance of thefirst wavelength of light, it may emit light at a second wavelength. Inan embodiment, a sample mixed with a reference particle may be exposedto a wavelength of light capable of exciting a fluorescent molecule in areference particle of interest and emitted light from the fluorescentmolecule may be measured. Specific fluorescence from a referenceparticle may be used to detect reference particles, and information fromdetected reference particles in a sample may be used for autofocusing.

Reference particles may be of any shape, such as spherical or cuboid.Reference particles include, without limitation, beads and microspheres.Reference particles may be of any size, such as with a diameter orlength of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 μm. Referenceparticles may be made of, or may contain, any suitable material, such aspolystyrene, polyethylene, latex, acrylic, or glass. For example, areference particle may be a polystyrene bead, e.g., a polystyrene beadhaving a diameter of between about 0.1 μm and about 50 μm; or betweenabout 1 μm and about 20 μm; or between about 5 μm and about 15 μm; orhaving a diameter of about 10 μm.

In one embodiment, a method for focusing a microscope is provided, whichmay include one or more of the following steps. First, a samplecontaining an object for microscopic analysis (e.g. bacteria,erythrocytes, etc.) may be mixed with a reference particle. Thereference particle may contain or be labeled with a molecule tofacilitate the detection of the particle, such as a fluorophore. Second,the mixture containing the reference particle and the sample may bepositioned into a light path of a microscope, for example in cuvette orslide. Optionally, the reference particle may sink to the bottom of thesample in the cuvette or slide, such that the reference particle restson the lowest surface of the cuvette or slide which is in contact withthe sample. The microscope may be of any type, including a fluorescentmicroscope. Third, the mixture may be exposed to a light beam configuredto visualize the reference particle. The light beam may be of any type,and may be of any orientation relative to the reference particle. Forexample, the light beam may be at a wavelength capable of exciting afluorophore within or attached to the reference particle. Exposure ofthe reference particle to the light beam may result in, for example, thegeneration and emission of light at a particular wavelength from thereference particle or scattering of light from the reference particle.Fourth, light emitted or scattered from the reference particle may bedetected by the microscope, and this information may be used in order todetermine the position of the reference particle within the mixture orto focus the microscope. Optionally, the microscope may be focused intoa plane of focus suited for objects of similar size to the referenceparticle. An image from the microscope may be obtained by an imagesensor. The image may be saved or or used for image analysis.

In some embodiments, a plurality of reference particles may be added toa sample. The reference particles may be all of the same size, or theymay be of different sizes. In some embodiments, reference particles ofdifferent sizes contain different fluorophores. Different fluorophoresmay have different absorption wavelengths, different emissionwavelengths, or both.

In an embodiment, a method for focusing a microscope is provided,including mixing more than one reference particle of known size with asample for microscopy, wherein at least two of the reference particlesare of different sizes and contain different fluorophores. The methodmay include one or more of the following steps. First, a samplecontaining an object for microscopic analysis may be mixed with two ormore reference particles, wherein at least two of the referenceparticles are of different sizes and contain different fluorophores(i.e. the “first reference particle” and the “second referenceparticle”). Second, the mixture containing the reference particles andthe sample may be positioned into the light path of a microscope. Themicroscope may be of any type, including a fluorescent microscope.Third, the mixture may be exposed to a light beam configured tovisualize the first reference particle. The light beam may be of anytype, and may be of any orientation relative to the first referenceparticle. For example, the light beam may be at a wavelength capable ofexciting a fluorophore within or attached to the first referenceparticle. Exposure of the first reference particle to the light beam mayresult in the generation and emission or scattering of light at aparticular wavelength from the first reference particle. Fourth, lightemitted or scattered from the first reference particle may be detected,and this information may be used in order to determine the position ofthe first reference particle within the mixture or to focus themicroscope into a first plane of focus suited for objects of similarsize to the first reference particle. Optionally, an image of the firstfocal plane may be obtained by an image sensor. The image may be savedor or used for image analysis. Fifth, the mixture may be exposed to alight beam configured to visualize the second reference particle. Thelight beam may be of any type, and may be of any orientation relative tothe second reference particle. Exposure of the second reference particleto the light beam may result in the generation and emission orscattering of light at a particular wavelength from the second referenceparticle. Sixth, light emitted or scattered from the second referenceparticle may be detected, and this information may be used in order todetermine the position of the second reference particle within themixture or to focus the microscope into a second plane of focus suitedfor objects of similar size to the second reference particle.Optionally, an image of the second focal plane may be obtained by animage sensor. The image may be saved or or used for image analysis.

In other embodiments, provided herein are systems and devices forcontext-based microscopy autofocus. The systems and devices may contain,without limitation, a fluorescence microscope. In an embodiment, thesystems and devices may be automated to add a reference particle havinga known size to a sample for microscopic analysis to form a mixture, toposition the mixture into the light path of a microscope, to expose themixture to a light beam configured to visualize the reference particle,to determine the position of the reference particle within the mixtureor to focus the microscope based on the position of the referenceparticle within the mixture. In an embodiment, a system or method forcontext-based microscopy autofocus may be configured as described inU.S. patent application Ser. No. 13/244,947 or Ser. No. 13/355,458,which are hereby incorporated by reference in their entireties.

Locating a Sample Holder

In some embodiments, methods, systems, and devices are provided hereinfor determining the location of a sample holder, or of a portion of, orindicial mark on, a sample holder. Such a determination is preferably aprecise determination, and is useful for identifying cells, particles,or other objects in a field of view within a sample holder even after asample holder has been moved, or a field of view has been altered (e.g.,by changing focus, or by inspection of different areas in a sampleholder).

In embodiments, an image based feedback mechanism may be used toaccurately and precisely determine a certain location in a cuvette,e.g., in a channel or other region containing a sample (see, e.g., ananalysis area 608 shown in FIGS. 7 and 8). Such determination,particularly when the sample holder is moved, and then returned to aprevious position, is important for comparison of images and opticalmeasurements taken before such movement, and after such movement.Variability from multiple sources may affect the position of the samplerelative to the axis of the imaging system; for example, variability incuvette parts, variability in cuvette assembly, variability in cuvettepositioning on the imaging system, and other possible sources ofvariability may affect the position of a sample with respect to theimaging system even if the sample remains in the same position withinthe sample holder. Methods for identifying and characterizing theposition of a sample holder with respect to an imaging system aredisclosed herein. For example, in order to accurately and reproduciblyimage an area of interest in a cuvette, a cuvette registration programmay be run. In embodiments, such a program begins by analyzing imagestaken at a predefined location in a sample holder, the predefinedlocation being close to a registration feature or fiducial marker withinthe field of view, or otherwise detectable by the program. A cuvetteregistration program comprises an image processing program, which imageprocessing program searches for the existence of the fiducial marker inthe image and returns either a yes/no answer (regarding whether or notthe fiducial marker is found within the inspected region) or aprobability of the marker being in the image. In instances where thefiducial marker is not found in the area that has been inspected, asearch algorithm is then used, which moves the area of inspection to adifferent location on or in the sample holder, and repeats the imaging.This process is repeated until the program finds the fiducial marker(i.e. gets a “yes” to the question of whether or not the fiducial markeris found within the inspected region, or maximizes the probability ofthe marker being within that region). Once the position of the fiducialmarker is identified, all other positions in or on the sample holder maybe determined, since the dimensions and layout of the sample holder areknown. Thus, following identification of the location of the fiducialmarker, any point of interest for imaging can be found and imaged, asthe location of the point of interest is thus known also (i.e., itsdistance and orientation from the fiducial marker is known, and, sincethe position of the fiducial marker is known, the point of interest isalso known). In embodiments, a fiducial marker can be or include aspecially engineered feature on the cuvette itself (e.g., may be a hole,a protrusion, a printed or molded pattern, or other feature) which canbe manufactured to be in the same location for every part to any desiredtolerance. In embodiments, a fiducial marker may be or include a featureof the cuvette (e.g., the edge of a channel) that is always at a fixeddistance from the point of interest (e.g., where the fiducial marker isthe edge of channel, the fiducial marker is always a fixed distance fromthe central axis of the channel).

Cell Counting/Enumerating Cells

In some embodiments, methods, systems, and devices are provided hereinfor enumerating cells in a sample.

Certain traditional methods for staining cell-containing samples involvestaining a specific volume of a sample (e.g. blood) with a particularconcentration or amount of stain. This may be referred to as “volumetricstaining” Volumetric staining has a number of shortcomings, including:(i) it fails to address normal variations in cell subpopulations betweendifferent subjects (e.g. different healthy subjects may have widelydifferent numbers of subpopulations of cells, such as CD3+ T cells(where “CD3+” indicates that the T cells express the CD3 marker)) and(ii) it fails to address that pathological samples may have dramaticallydifferent cellular composition when compared to healthy samples (e.g.the percent and number of CD3+ T cells in blood are greatly elevated inpatients with T cell leukemia over the percent and number in healthysubjects).

For accurate and reproducible staining of cell-containing samples, itmay be desirable to add a specific amount of a cellular stain (e.g. DNAdyes, antibodies, binders, etc.) to a specific number or concentrationof cells. For example, it may be desirable to add 0.2 micrograms of aparticular stain for white blood cells per 1000 white blood cells in asample. After an incubation period of the dye with the cells, a samplemay be washed to remove excess (unbound) dye, prepared to an appropriatecell density for microscopy, and imaged. In this manner, a stain andstaining procedure can be optimized or normalized for a particular cellnumber.

In one embodiment, a method is provided for enumerating the number ofcells of interest in a sample. The method may include one or more of thefollowing steps or elements. A first stain that will bind to the cellsof interest in a sample may be added to the sample. The mixture of firststain and sample may be incubated. The cells in the mixture of firststain and sample may be washed to remove excess (unbound) stain. Thewashed cells stained with a first stain may be prepared in a desiredvolume for further analysis. The washed cells stained with a first stainmay be analyzed by a spectrophotometer. Data from the spectrophotometermay be used to enumerate the approximate number of cells in the sample.For example, the first stain may be a fluorescent dye which binds tonucleic acids, and the spectrophotometer may include a light sourcewhich emits light at an excitation wavelength of the fluorescent dye,and a light sensor which can detect light in the emission wavelength ofthe fluorescent dye. In this example, based on the fluorescent signalfrom the dye, the approximate amount of nucleic acid in the sample maybe calculated, and from this approximate amount of nucleic acid in thesample, the approximate number of cells in the sample may be determined.Based on the number of cells in the sample, a second stain that willbind to cells of interest in a sample may be added to the sample. Inembodiments, the amount of second stain added to the sample may bedetermined in view of the approximate number of cells determined usingthe first stain. In embodiments, the amount of second stain added to thesample may be calculated using the number of cells determined by use ofthe first stain, in order that a desired ratio of second stain per cellbe obtained. The mixture of second stain and sample may be incubated.The cells in the mixture of second stain and sample may be washed toremove excess stain. The washed cells stained with a second stain may beprepared in a desired volume for further analysis. The washed cellsstained with a second stain may be analyzed by microscopy.

Enumerating Cells in a Sample Prior to Determining the Ploidy of Cells

In one embodiment, a method for enumerating cells in a sample prior todetermining the ploidy of the cells is provided, wherein the methodincludes one or more of the following steps or elements. A first stainwhich binds to the cells of interest in the sample and that isspectrally distinct from the emission of a DNA dye may be added to thesample. The cells of interest may be, for example, white blood cells.The first stain may be, for example, a fluorophore-conjugated antibody.A fluorophore-conjugated antibody may bind to, for example, a widelyexpressed antigen (e.g. CD45), or it may bind to an antigen expressed bya specific sub-population of cells (e.g. CD3 for T cells). The mixtureof first stain and sample may be incubated. The cells in the mixture offirst stain and sample may be washed to remove excess (unbound) stain.The washed cells stained with a first stain may be prepared in a desiredvolume for further analysis. The washed cells stained with a first stainmay be analyzed by a spectrophotometer. Data from the spectrophotometermay be used to enumerate the approximate number of cells in the sample.Based on the number of cells in the sample, a second stain that willbind to cells of interest in a sample may be added to the sample. Thesecond stain may be a DNA dye, such as propidium iodide or4′,6-diamidino-2-phenylindole (“DAPI”). In embodiments, the amount ofsecond stain added to the sample may be determined in view of theapproximate number of cells determined using the first stain. Inembodiments, the amount of second stain added to the sample may becalculated using the number of cells determined by use of the firststain, in order that a desired ratio of second stain per cell beobtained. The mixture of second stain and sample may be incubated. Thecells in the mixture of second stain and sample may be washed to removeexcess stain. The washed cells stained with a second stain may beprepared in a desired volume for further analysis. The washed cellsstained with a second stain may be analyzed for ploidy by microscopy.

In methods for determining the ploidy of cells, it may be important tocombine a given number of cells for ploidy analysis with a certainamount or concentration of DNA stain, in order to generate accurate andconsistent data regarding the ploidy of the cells. In one example, thenumber of white blood cells per volume of blood may vary within ahealthy population, and thus, it may be desirable to determine thenumber of white blood cells in a volume of blood before attempting tostain the white blood cells for ploidy analysis.

The methods provided above for determining the ploidy of cells may alsobe performed for any method in which enumerating cells in a sample priorto determining an attribute related to the nucleic acid content of acell is desired. For example, the above method may be used with methodsinvolving enumerating cells in a sample prior to determining themorphology of nuclei of cells, the size of the nuclei of cells, theratio of nuclei area to total cell area, etc.

Enumerating Cells in a Sample Prior to Cell Surface Staining

In one embodiment, a method for enumerating cells in a sample prior tocell surface staining is provided, wherein the method includes one ormore of the following steps or elements. A first stain which binds tothe cells of interest in the sample and that is spectrally distinct fromthe emission of a dye to be used to stain the surface of the cells ofinterest may be added to the sample. The cells of interest may be, forexample, white blood cells. The first stain may be, for example, a DNAdye (e.g. propidium iodide, DRAQ5® or DAPI). The mixture of first stainand sample may be incubated. The cells in the mixture of first stain andsample may be washed to remove excess (unbound) stain. The washed cellsstained with a first stain may be prepared in a desired volume forfurther analysis. The washed cells stained with a first stain may beanalyzed by a spectrophotometer. Data from the spectrophotometer may beused to enumerate the approximate number of cells in the sample. Basedon the number of cells in the sample, a second stain that will bind tocells of interest in a sample may be added to the sample. Inembodiments, the amount of second stain added to the sample may bedetermined in view of the approximate number of cells determined usingthe first stain. In embodiments, the amount of second stain added to thesample may be calculated using the number of cells determined by use ofthe first stain, in order that a desired ratio of second stain per cellbe obtained. The second stain may be, for example, afluorophore-conjugated antibody. A fluorophore-conjugated antibody maybind to, for example, a widely expressed antigen (e.g. CD45), or it maybind to an antigen expressed by a specific sub-population of cells (e.g.CD3 for T cells). The mixture of second stain and sample may beincubated. The cells in the mixture of second stain and sample may bewashed to remove excess stain. The washed cells stained with a secondstain may be prepared in a desired volume for further analysis. Thewashed cells stained with a second stain may be analyzed for a cellsurface antigen by microscopy.

In methods for cell surface antigen staining of cells, it may beimportant to combine a given number of cells for analysis with a certainamount or concentration of cell surface antigen stain, in order togenerate accurate and consistent data regarding the content of the cellsurfaces. In one example, the number of white blood cells per volume ofblood may vary within a healthy population (blood from healthy subjectstypically has between about 3000 and 10,000 WBCs per microliter (μL)),and thus, it may be desirable to determine the number of white bloodcells in a volume of blood before attempting to stain the white bloodcells for cell surface antigens. In another example, the number of whiteblood cells per volume of blood may vary between healthy and sicksubjects (e.g., lymphoma patients may have up to 100,000 WBCs per μL ofblood), and thus, it may be desirable to determine the number of whiteblood cells in a volume of blood before attempting to stain the whiteblood cells for cell surface antigens.

Thus, as a theoretical example, a healthy patient may have 5000 cellsper μL of blood, and 500 of these are CD3+ T cells, while a lymphomapatient may have 50,000 cells per microliter of blood and 45,000 ofthese are CD3+ T cells. If 100 microliters of blood is traditionallystained, then a sample from a healthy subject would contain about500,000 total cells, of which about 50,000 cells would be CD3+ T cells.A 100 microliter sample from a lymphoma subject would contain about5,000,000 total cells, of which about 4,500,000 cells would be CD3+ Tcells. In this theoretical example, the pathological sample contains tentimes the number of total cells and ninety times the number of CD3+ Tcells, when compared to a sample from a healthy subject. If thepathological sample would be stained with a traditional “volumetricstaining” approach that is optimized for samples from healthy subjects,the sample from the lymphoma subject may be insufficiently stained. Forthis reason, for example, the present methods in which a prior estimateof the number of cells in a sample is used to adjust the amount of dyeapplied to a sample provide advantages over traditional volumetricstaining methods.

Accordingly, methods provided herein may be used to enumerate cells in asample before cell staining, in order to generate accurate or consistentdata regarding samples.

Method Speeds

Methods, systems, and devices provided herein may support the rapidacquisition of sample analysis results. Methods provided herein mayprovide analysis results in less than, for example, about 6 hours, 4hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10minutes, or 5 minutes from the initiation of the method.

Rapid analysis results may be used to provide real-time informationrelevant to the treatment, diagnosis, or monitoring of a patient. Forexample, rapid analysis results may be used to guide a treatmentdecision of a surgeon operating on a patient. During surgery, a surgeonmay obtain a biological sample from a patient for analysis. By receivingrapid analysis of a sample by a method provided herein, a surgeon may beable to make a treatment decision during the course of surgery.

In another example, rapid analysis results provided by the methods,systems, and devices provided herein may support a patient receivinginformation regarding a biological sample provided by the patient at apoint of service during the same visit to the point of service locationin which the patient provided the biological sample.

For example, Applicants describe herein a rapid assay which may be usedto prepare a sample of whole blood for analysis of white blood cells forthe presence of multiple markers and cell types. Such an assay is usefulfor preparing samples of whole blood for imaging analysis; the samplesare ready for imaging in less than about 20 minutes, or in less thanabout 15 minutes.

Rapid White Blood Cell Assay from Whole Blood

This assay prepares samples of whole blood for cytometric analysis ofwhite blood cells in less than about 15 minutes or less than about 20minutes. Automated cytometric analysis of such prepared cells may alsobe done rapidly, so that cytometric WBC analysis can be performed fromwhole blood in about half an hour or less. In addition, this assay usesonly a small volume of the blood sample, so is sparing of resources, andless inconvenient or uncomfortable to a subject than assays whichrequire larger volumes of blood.

Reagents used in this assay include: phosphate buffered saline, Lyse Fixbuffer, beads, resuspension buffer, and reagent cocktails which containdyes and dye-conjugated antibodies. The antibodies are directed tospecific WBC markers.

Phosphate buffered saline (PBS): 137 mM NaCl, 3 mM KCl, 8 mM, Na2HPO4,1.5 mM KH2PO4, pH adjusted to pH to 7.2 to pH 7.4 (with HCl).

Resuspension buffer (RSB): 5% bovine serum albumin in PBS.

Lyse Fix buffer: 0.0266% saponin in PBS with 10% paraformaldehyde (PFA),where “%” indicates grams/100 mL (final ratio is approximately 13:1saponin PBS:PFA).

Reagent Cocktail 1: DRAQ5®, anti-CD14 antibody conjugated to PacificBlue™ dye, Fc block (e.g., immunoglobulin such as mouse IgG), in 0.2%BSA in PBS.

Reagent Cocktail 2: anti-CD16 antibody conjugated to phycoerythrin (PE)dye, anti-CD45 antibody conjugated to Alexa Fluor® 647 dye, anti-CD123antibody conjugated to PECy5 dye, Fc block (e.g., immunoglobulin), in15% BSA in PBS.

Assay steps include:

Obtain whole blood from a subject.

Place 50 μL of whole blood in a tube. If desired, the blood sample maybe acquired directly to a tube. Where 50 μL is the total amount of bloodtaken from the subject, then the entire sample is added or acquired to atube; where more than 50 μL is acquired from a subject, then the 50 μLis an aliquot of the sample.

Centrifuge the sample at 1200×g for 3 minutes.

Remove 20 μL of plasma from the tube.

Place the tube on heat block (to raise the temperature to 37° C.), add20 μL of RSB, and mix thoroughly.

Add Cocktail 1 (approximately 5 μL). (In embodiments, Cocktail 1 may beadded directly to whole blood, and the previous steps of centrifugation,removal of an aliquot of plasma and replacement with RSB may beomitted.)

Incubate the sample at 37° C. for 2 minutes.

Add Lyse Fix buffer (at a 6:1 ratio of (Lyse Fix buffer) to (stainedblood); approximately 300-350 μL). A known concentration of beads may beincluded in the Lyse Fix buffer to provide targets (reference particles)for focusing and to provide a calibration for the concentration of thesample (e.g., as described above under the heading “Context-basedAutofocus”). Polystyrene or other beads, having diameters of about 1micron to about 30 microns, may be used. For example, 10 micronpolystyrene beads at a concentration of about 100 beads to about 2000beads per microliter (μL), or of about 150 beads to about 1500 beads perμL, or of about 200 beads to about 1000 beads per μL, may be used.

Incubate in the Lyse Fix buffer at 37° C. for a total of 3 minutes; atabout 1.5 minutes after addition of the buffer, mix by pipetting thesolution up and down five times.

Centrifuge the sample mixture at 1200×g for 3 minutes.

Remove the supernatant (approximately 350 μL). Save the supernatant toadjust the volume, if needed, in later steps.

Add Cocktail 2 (approximately 15 μL) to provide the final mixture.

Load the final mixture on a pre-warmed imaging cuvette (37° C.).

Incubate the cuvette at 37° C. for 5 minutes before imaging.

Image the sample.

Thus, the sample is ready for imaging in less than about 15 minutes. Inembodiments, some of the steps may be shortened (e.g., in alternativeembodiments, a centrifugation step or an incubation step may beshortened). Since the methods disclosed above prepare the sample usingcocktails which include multiple dyes, analysis of these samples for thepresence of several cell-type markers may be performed within a singlefield of view, providing efficient imaging of the samples with minimalduplication of effort. Light scatter images of these same fields of viewprovides yet another aspect of analysis which may be applied efficientlywithout requiring separate samples or separate fields of view for theseveral modes of image analysis of the samples. Inclusion of referenceparticles of a known size further aids imaging by allowing use ofautomatic focusing and, since the concentration of the referenceparticles is known, provides an independent measure of sample dilutionand cell concentration in each image.

The imaging of the prepared sample may also be done rapidly; forexample, such imaging may be performed in about 10 minutes (typicallybetween about 2 minutes and about 12 minutes) by automatic deviceshaving features as described herein and, for example, in U.S. patentapplication Ser. No. 13/244,947, in U.S. patent application Ser. No.13/769,779, and related applications. Thus, in embodiments, the entireanalysis, including preparation of the blood sample and imaging of theprepared sample, may be performed in about 30 minutes or less.

The images and image analysis obtained from samples prepared accordingto the methods discussed above (and similar methods discussed below) aresuitable for identifying different populations of WBCs from whole blood.Such identification and quantification is done rapidly on the samesample by illumination of the sample (e.g., sequentially) with differentwavelengths of light and recording and analyzing the resulting imagesand light intensities. Such methods are suitable for providing theimages and plots as shown, for example, in FIGS. 9A-9F, 10, and 11,which were prepared using methods as disclosed herein (e.g., methodsdiscussed both supra and infra). The comparisons shown in FIG. 12demonstrate that these methods are accurate and reliable, and correlatewell with other methods (e.g., analysis by an Abbott CELL-DYN RubySystem (Abbott Diagnostics, Lake Forest, Ill., USA)) the referenceanalyzer used for the comparisons shown in FIG. 12.

Analysis of Pathology Samples

Any of the methods provided herein may be used to analyzecell-containing pathology samples. If a pathology sample is a tissuesample, the sample may be treated to separate the cells of the tissueinto individual cells for analysis by methods provided herein.

Analysis of pathology samples by any of the methods provided herein maysupport rapid pathology analysis, and the rapid integration of pathologyanalysis results into a treatment decision for a patient.

Additional Procedures in Response to Analysis Results

In some embodiments, the devices and systems provided herein may beconfigured to trigger an additional procedure in response to a resultobtained by an analysis method provided herein.

In one example, a device or system may be programmed to provide an alertto a user if a result is outside of an expected range. The alert mayprompt a user or medical personnel to, for example, manually analyze asample, check the device or system for proper operation, etc.

In another example, a device or system may be programmed toautomatically run one or more additional tests on a sample if a resultis within or outside of a certain range. In some examples, devices andsystems provided herein are capable of performing multiple differentassays, and the device or system may run an addition assay to verify orfurther investigate a result generated by a method provided herein.

Analysis Using Non-Specific Dyes

One non-limiting example to accelerate imaging is to use a “high light”situation, where cells are labeled with very high concentration of dyes.In the present embodiment, non-specific dyes are used that label theDNA, the membranes, or other portion of the cells. This example does notuse antibody dyes that target specific and rare proteins or othermarkers.

With the non-specific dye, it is possible to obtain cell informationwithout requiring a separation step (such as, e.g., separation bycentrifugation or by performing physical separation). Without thisseparation step, one can more rapidly move directly to imaging thesample, such as but not limited imaging a large area of cells that mayinclude both a) non-target cells such as red blood cells (RBCs) and b)target cells or objects of interest such as white blood cells (WBCs).Thus, in one non-limiting example of imaging a blood sample, one canimage five million RBCs and five thousand or other number of WBCstherein. The targeted cells can be differentiated based on what isinside the cell such as but not limited to the shape of the nucleus of acell. In one embodiment, a nuclear stain is used to stain the nuclei ofcells in a sample, and based on the kind and amount of staining aparticular cell has (e.g., the presence of nuclear staining, or theshape of a stained nucleus, or other characteristic), one can determineits cell type based on this staining, even though the dye isnon-specific. In other examples, other internal shapes in the cell (suchas, e.g., whether or not the cytoplasm has granules or other objectstherein) can be indicative or characteristic and be used to identify andquantify cells in a sample. For a urine sample, any cells present, andcrystal shapes in the sample can be used to identify a sample and todetermine whether or not abnormalities are found. In this manner, theuse of non-specific dyes can be used to rapidly image cells in a mannerthat can be used to determine cells as desired.

Analysis Using a Plurality of Excitation or Detection Channels

In the context of using even smaller sample volumes for cytometry, inembodiments of advanced cytometry assays, an additional excitation ordetection wavelength may be used. For example, for classification ofWBCs in a lymphocyte subset assay, the various cells such as T cells, Bcells, K cells, and other cells are to be counted. In this case, oneuses two markers merely to identify that the cell is a lymphocyte. Tofurther sub-classify the cells in a blood sample, for example, one mayagain use two markers. Thus, if one has a system that can only detecttwo colors at a time, there is an insufficient number of wavelengths forthe analysis.

In one embodiment, one can aliquot the sample to make two separatesample portions and then one can image one combination in one part andanother combination in another part of the system, using different partsof the sample. Unfortunately, this can cause a doubling of volume andtime. The more independent channels that are built into a system, thelesser the number of these sample parts or volume used.

EXAMPLES Cell Processing

In embodiments, it is often useful to process biological samples forimaging, testing, and analysis. For example, it is often useful toprocess biological samples containing cells for imaging, testing, andanalysis.

Processing of a biological sample may include pre-processing (e.g.,preparation of a sample for a subsequent processing or measurement),processing (e.g., alteration of a sample so that it differs from itsoriginal, or previous, state), and post-processing (e.g., disposal ofall or a portion of a sample following its measurement or use). Abiological sample may be divided into portions, such as aliquots of ablood or urine sample, or such as slicing, mincing, or dividing a tissuesample into two or more pieces. Processing of a biological sample, suchas blood sample, may include mixing, stirring, sonication,homogenization, or other processing of a sample or of a portion of thesample. Processing of a biological sample, such as blood sample, mayinclude centrifugation of a sample or a portion thereof. Processing of abiological sample, such as blood sample, may include providing time forcomponents of the sample to separate or settle, and may includefiltration (e.g., passing the sample or a portion thereof through afilter). Processing of a biological sample, such as blood sample, mayinclude allowing or causing a blood sample to coagulate. Processing of abiological sample, such as blood sample, may include concentration ofthe sample, or of a portion of the sample (e.g., by sedimentation orcentrifugation of a blood sample, or of a solution containing ahomogenate of tissue from a tissue sample) to provide a pellet and asupernatant. Processing of a biological sample, such as blood sample,may include dilution of a portion of the sample. Dilution may be of asample, or of a portion of a sample, including dilution of a pellet orof a supernatant from sample. A biological sample may be diluted withwater, or with a saline solution, such as a buffered saline solution. Abiological sample may be diluted with a solution which may or may notinclude a fixative (e.g., formaldehyde, paraformaldehyde,glutaraldehyde, or other cross-linking agent). A biological sample maybe diluted with a solution effective that an osmotic gradient isproduced between the surrounding solution and the interior, or aninterior compartment, of such cells, effective that the cell volume isaltered. For example, where the resulting solution concentrationfollowing dilution is less than the effective concentration of theinterior of a cell, or of an interior cell compartment, the volume ofsuch a cell will increase (i.e., the cell will swell). A biologicalsample may be diluted with a solution which may or may not include anosmoticant (such as, for example, glucose, sucrose, or other sugar;salts such as sodium, potassium, ammonium, or other salt; or otherosmotically active compound or ingredient). In embodiments, anosmoticant may be effective to maintain the integrity of cells in thesample, by, for example, stabilizing or reducing possible osmoticgradients between the surrounding solution and the interior, or aninterior compartment, of such cells. In embodiments, an osmoticant maybe effective to provide or to increase osmotic gradients between thesurrounding solution and the interior, or an interior compartment, ofsuch cells, effective that the cells at least partially collapse (wherethe cellular interior or an interior compartment is less concentratedthan the surrounding solution), or effective that the cells swell (wherethe cellular interior or an interior compartment is more concentratedthan the surrounding solution).

A biological sample may be contacted with a solution containing asurfactant, which may disrupt the membranes of cells in the sample, orhave other effects on cell morphology. For example, contacting RBCs witha low concentration of a surfactant causes the RBCs to lose theirdisc-like shape and to assume a more spherical shape.

A biological sample may be dyed, or markers may be added to the sample,or the sample may be otherwise prepared for detection, visualization, orquantification of the sample, a portion of a sample, a component part ofa sample, or a portion of a cell or structure within a sample. Forexample, a biological sample may be contacted with a solution containinga dye. A dye may stain or otherwise make visible a cell, or a portion ofa cell, or a material or molecule associated with a cell in a sample. Adye may bind to or be altered by an element, compound, or othercomponent of a sample; for example a dye may change color, or otherwisealter one of more of its properties, including its optical properties,in response to a change or differential in the pH of a solution in whichit is present; a dye may change color, or otherwise alter one of more ofits properties, including its optical properties, in response to achange or differential in the concentration of an element or compound(e.g., sodium, calcium, CO₂, glucose, or other ion, element, orcompound) present in a solution in which the dye is present. Forexample, a biological sample may be contacted with a solution containingan antibody or an antibody fragment. For example, a biological samplemay be contacted with a solution that includes particles. Particlesadded to a biological sample may serve as standards (e.g., may serve assize standards, where the size or size distribution of the particles isknown, or as concentration standards, where the number, amount, orconcentration of the particles is known), or may serve as markers (e.g.,where the particles bind or adhere to particular cells or types ofcells, to particular cell markers or cellular compartments, or where theparticles bind to all cells in a sample).

Cytometry includes observations and measurements of cells, such as redblood cells, platelets, white blood cells, including qualitative andquantitative observations and measurements of cell numbers, cell types,cell surface markers, internal cellular markers, and othercharacteristics of cells of interest. Where a biological sample includesor is a blood sample, the sample may be divided into portions, and maybe diluted (e.g., to provide greater volume for ease of handling, toalter the density or concentration of cellular components in the sampleto provide a desired diluted density, concentration, or cell number orrange of these, etc.). The sample may be treated with agents whichaffect coagulation, or may be treated or handled so as to concentrate orprecipitate sample components (e.g., ethylene diamine tetraacetic acid(EDTA) or heparin may be added to the sample, or the sample may becentrifuged or cells allowed to settle). A sample, or portion of asample, may be treated by adding dyes or other reagents which may reactwith and mark particular cells or particular cellular components. Forexample, dyes which mark cell nuclei (e.g., hematoxylin dyes, cyaninedyes, drag dyes such as DRAQ5®, and others); dyes which mark cellcytoplasm (e.g., eosin dyes, including fluorescein dyes, and others) maybe used separately or together to aid in visualization, identification,and quantification of cells. More specific markers, including antibodiesand antibody fragments specific for cellular targets, such as cellsurface proteins, intracellular proteins and compartments, and othertargets, are also useful in cytometry.

Biological samples may be measured and analyzed by cytometry usingoptical means, including, for example, photodiode detectors,photomultipliers, charge-coupled devices, laser diodes,spectrophotometers, cameras, microscopes, or other devices which measurelight intensity (of a single wavelength, of multiple wavelengths, or ofa range, or ranges, of wavelengths of light), form an image, or both. Afield of view including a sample, or portion of a sample, may be imaged,or may be scanned, or both, using such detectors. A biological samplemay be measured and analyzed by cytometry prior to processing, dilution,separation, centrifugation, coagulation, or other alteration. Abiological sample may be measured and analyzed by cytometry during orfollowing processing, dilution, separation, centrifugation, coagulation,or other alteration of the sample. For example, a biological sample maybe measured and analyzed by cytometry directly following receipt of thesample. In other examples, a biological sample may be measured andanalyzed by cytometry during or after processing, dilution, separation,centrifugation, coagulation, or other alteration of the sample.

For example, a blood sample or portion thereof may be prepared forcytometry by sedimentation or centrifugation. A sedimented or pelletportion of such a sample may be resuspended in a buffer of choice priorto cytometric analysis (e.g., by aspiration, stirring, sonication, orother processing). A biological sample may be diluted or resuspendedwith water, or with a saline solution, such as a buffered salinesolution prior to cytometric analysis. A solution used for such dilutionor resuspension may or may not include a fixative (e.g., formaldehyde,paraformaldehyde, or other agent which cross-links proteins). A solutionused for such dilution or resuspension may provide an osmotic gradientbetween the surrounding solution and the interior, or an interiorcompartment, of cells in the sample, effective that the cell volume ofsome or all cells in the sample is altered. For example, where theresulting solution concentration following dilution is less than theeffective concentration of the interior of a cell, or of an interiorcell compartment, the volume of such a cell will increase (i.e., thecell will swell). A biological sample may be diluted with a solutionwhich may or may not include an osmoticant (such as, for example,glucose, sucrose, or other sugar; salts such as sodium, potassium,ammonium, or other salt; or other osmotically active compound oringredient). In embodiments, an osmoticant may be effective to maintainthe integrity of cells in the sample, by, for example, stabilizing orreducing possible osmotic gradients between the surrounding solution andthe interior, or an interior compartment, of such cells. In embodiments,an osmoticant may be effective to provide or to increase osmoticgradients between the surrounding solution and the interior, or aninterior compartment, of such cells, effective that the cells at leastpartially collapse (where the cellular interior or an interiorcompartment is less concentrated than the surrounding solution), oreffective that the cells swell (where the cellular interior or aninterior compartment is more concentrated than the surroundingsolution).

For example, a biological sample may be measured or analyzed followingdilution of a portion of the sample with a solution including dyes. Forexample, a biological sample may be measured or analyzed followingdilution of a portion of the sample with a solution including antibodiesor antibody fragments. For example, a biological sample may be measuredor analyzed following dilution of a portion of the sample with asolution including particles. Particles added to a biological sample mayserve as standards (e.g., may serve as size standards, where the size orsize distribution of the particles is known, or as concentrationstandards, where the number, amount, or concentration of the particlesis known), or may serve as markers (e.g., where the particles bind oradhere to particular cells or types of cells, to particular cell markersor cellular compartments, or where the particles bind to all cells in asample).

For example, a biological sample may be measured or analyzed followingprocessing which may separate one or more types of cells from anothercell type or types. Such separation may be accomplished by gravity(e.g., sedimentation); centrifugation; filtration; contact with asubstrate (e.g., a surface, such as a wall or a bead, containingantibodies, lectins, or other components which may bind or adhere to onecell type in preference to another cell type); or other means.Separation may be aided or accomplished by alteration of a cell type ortypes. For example, a solution may be added to a biological sample, suchas a blood sample, which causes some or all cells in the sample toswell. Where one type of cell swells faster than another type or typesof cell, cell types may be differentiated by observing or measuring thesample following addition of the solution. Such observations andmeasurements may be made at a time, or at multiple times, selected so asto accentuate the differences in response (e.g., size, volume, internalconcentration, or other property affected by such swelling) and so toincrease the sensitivity and accuracy of the observations andmeasurements. In some instances, a type or types of cells may burst inresponse to such swelling, allowing for improved observations andmeasurements of the remaining cell type or types in the sample.

Observation, measurement and analysis of a biological sample bycytometry may include photometric measurements, for example, using aphotodiode, a photomultiplier, a laser diode, a spectrophotometer, acharge-coupled device, a camera, a microscope, or other means or device.Cytometry may include preparing and analyzing images of cells in abiological sample (e.g., two-dimensional images), where the cells arelabeled (e.g., with fluorescent, chemiluminescent, enzymatic, or otherlabels) and plated (e.g., allowed to settle on a substrate) and imagedby a camera. The camera may include a lens, and may be attached to orused in conjunction with a microscope. Cells may be identified in thetwo-dimensional images by their attached labels (e.g., from lightemitted by the labels).

An image of cells prepared and analyzed by a cytometer as disclosedherein may include no cells, one cell, or multiple cells. A cell or cellin an image of a cytometer, as disclosed herein, may be labeled, asdisclosed above. A cell or cell in an image of a cytometer, as disclosedherein, may be labeled, as disclosed above, effective to identify theimage, and the subject from whom the sample was taken.

In some embodiments, the assay system is configured to perform cytometryassays. Cytometry assays are typically used to optically, electrically,or acoustically measure characteristics of individual cells. For thepurposes of this disclosure, “cells” may encompass non-cellular samplesthat are generally of similar sizes to individual cells, including butnot limited to vesicles (such as liposomes), small groups of cells,virions, bacteria, protozoa, crystals, bodies formed by aggregation oflipids or proteins, and substances bound to small particles such asbeads or microspheres. Such characteristics include but are not limitedto size; shape; granularity; light scattering pattern (or opticalindicatrix); whether the cell membrane is intact; concentration,morphology and spatio-temporal distribution of internal cell contents,including but not limited to protein content, protein modifications,nucleic acid content, nucleic acid modifications, organelle content,nucleus structure, nucleus content, internal cell structure, contents ofinternal vesicles (including pH), ion concentrations, and presence ofother small molecules such as steroids or drugs; and cell surface (bothcellular membrane and cell wall) markers including proteins, lipids,carbohydrates, and modifications thereof. By using appropriate dyes,stains, or other labeling molecules either in pure form, conjugated withother molecules or immobilized in, or bound to nano- or micro-particles,cytometry may be used to determine the presence, quantity, ormodifications of specific proteins, nucleic acids, lipids,carbohydrates, or other molecules. Properties that may be measured bycytometry also include measures of cellular function or activity,including but not limited to phagocytosis, antigen presentation,cytokine secretion, changes in expression of internal and surfacemolecules, binding to other molecules or cells or substrates, activetransport of small molecules, mitosis or meiosis; protein translation,gene transcription, DNA replication, DNA repair, protein secretion,apoptosis, chemotaxis, mobility, adhesion, antioxidizing activity, RNAi,protein or nucleic acid degradation, drug responses, infectiousness, andthe activity of specific pathways or enzymes. Cytometry may also be usedto determine information about a population of cells, including but notlimited to cell counts, percent of total population, and variation inthe sample population for any of the characteristics described above.The assays described herein may be used to measure one or more of theabove characteristics for each cell, which may be advantageous todetermine correlations or other relationships between differentcharacteristics. The assays described herein may also be used toindependently measure multiple populations of cells, for example bylabeling a mixed cell population with antibodies specific for differentcell lines. A microscopy module may permit the performance of histology,pathology, or morphological analysis with the device, and alsofacilitates the evaluation of objects based on both physical andchemical characteristics. Tissues can be homogenized, washed, depositedon a cuvette or slide, dried, stained (such as with antibodies),incubated and then imaged. When combined with the data transmissiontechnologies described elsewhere herein, these innovations facilitatethe transmission of images from a CMOS/CDD or similar detector to, e.g.,a licensed pathologist for review, which is not possible withtraditional devices that only perform flow cytometry. The cytometer canmeasure surface antigens as well as cell morphology; surface antigensenable more sensitive and specific tesing compared to traditionalhematology laboratory devices. The interpretation of cellular assays maybe automated by gating of one or more measurements; the gatingthresholds may be set by an expert or learned based on statisticalmethods from training data; gating rules can be specific for individualsubjects or populations of subjects.

In some embodiments, the incorporation of a cytometer module into apoint of service device provides the measurement of cellular attributestypically measured by common laboratory devices and laboratories forinterpretation and review by classically-trained medical personnel,improving the speed or quality of clinical decision-making A point ofservice device may, therefore, be configured for cytometric analysis.

Example 1

A sample of cells containing blood leukocytes including natural killercells and neutrophils was obtained. The sample was treated with afluorescently labeled identity binder (anti-CD 16 binder), which bindsto both natural killer cells and neutrophils. The sample was alsotreated with a nuclear dye (DRAQ5®). The sample was imaged byfluorescence microscopy and dark field microscopy. The level offluorescence and light side scatter of different cells in the sample wasrecorded and analyzed. Segmented images containing the anti-CD16 bindersignal provided quantitative information on the fluorescence intensityof each cell (corresponding to the CD16 expression level), and also thesize of each cell. The dark field image provided quantitativeinformation on the scatter properties of each cell. Images containingthe DNA dye signal were segmented to determine the fluorescentintensity, size, and shape of the nucleus.

As shown in FIG. 1A, two major groupings cells were identified based onthe measurement of CD16 fluorescence and light scatter of the differentcells. The group of cells with bright/high CD16 fluorescence signal andhigh scatter (FIG. 1A, right circle) are neutrophils. The group of cellswith intermediate CD16 fluorescence signal and low scatter (FIG. 1A,left circle) are natural killer cells. While the measurement offluorescence and light scatter of the different cells provides enoughinformation to classify most cells in the sample as either naturalkiller cells or neutrophils, for some cells, measurement of theseattributes does not provide enough information to classify the cellswith a high degree of accuracy. For example, the measurement offluorescence and light scatter of cells does not provide enoughinformation to accurately classify the small group of cells in thesmallest circle in FIG. 1A (i.e. the middle circle). In order toidentify whether the cells in the smallest circle were natural killercells or neutrophils, images of the nuclear (DRAQ5®) and total cell(anti-CD16) staining of these were examined. Quantitative measurementsof the area of the nucleus and the total cell volume of the cells wereobtained, and the ratio of nuclear area to total cell area wasdetermined. As shown in FIG. 1B, there is a clear difference in theratio of nuclear area to total cell area between natural killer cells(“NK”) and neutrophils (“Neu”). Thus, the use of quantitative microscopyto examine multiple attributes of cells in the sample was used to allowfor unambiguous classification of cells. FIG. 1C shows images of naturalkiller cells from the smallest circle in FIG. 1A. All images have thesame length scale. The images on the left are cells stained for totalcell area (anti-CD16), and the images on the right are the same cellswith just nuclear staining (DRAQ5®). The images on the top and bottomrow are different examples of the natural killer cells. FIG. 1D showsimages of neutrophils from the smallest circle in FIG. 1A. All imageshave the same length scale. The images on the left are cells stained fortotal cell area, and the images on the right are the same cells withjust nuclear staining. The images on the top and bottom row aredifferent examples of the natural killer cells.

In addition, the nucleus of a neutrophil has a distinctive multi-lobedshape, whereas the nucleus of a natural killer cell (and otherlymphocytes) is round, even, and smooth. Image segmentation algorithmsmay be used to identify and classify cells based on the shape of thenucleus itself. Image segmentation is discussed further in Example 7below.

Example 2

A sample containing platelets was obtained. The platelets were labeledwith fluorescently conjugated anti-CD41 and anti-CD61 antibodies. Beadshaving a diameter of 3 μm were also added to the sample. The sample wasimaged at 10× and 20× magnifications (FIG. 2A). The intensity offluorescence distribution for individual platelets was measured (fromboth antibodies), and determined have a Gaussian shape (FIG. 2B). Themeasured values of fluorescence of individual platelets was plotted, anda fit for the intensity distribution was determined (FIG. 2C). In FIG.2C, the grey line is the measured fluorescence intensity across anindividual platelet, and the black line is the fit. Parameters of thefit, such as the mean of the Gaussian, the variance, the volume, thewidth, and the area of the base, etc., can be evaluated as predictors ofplatelet volume. The volume of the Gaussian and the width of the fithave been determined to correlate closely with mean platelet volume.

For the above measurements, the 3 μm beads served as references andfiducials for controlling variance in accurately determining the bestplane of focus, and the effect of this variance on the measurement ofvolume.

In addition, platelet size estimated based on fitting a 2D model can becalibrated to be in the normal range (FIG. 3).

Example 3

A sample containing red blood cells (“RBCs”) was obtained. The RBCs weretreated with a low concentration of a surfactant (DDAPS or SDS), causingthe RBCs to assume a sphere-like shape. The RBCs were imaged by darkfield microscopy in two different cuvettes: (A) a cuvette that allowedonly pure epi-illumination (FIG. 4A); and (B) a cuvette that allowed amixture of both epi and trans-illumination (FIG. 4B). The RBCs were muchmore visible in the cuvette that allowed a mixture of both epi andtrans-illumination over the cuvette that allowed only pureepi-illumination (FIGS. 4A-4B).

Example 4

A sample containing neutrophils was obtained. In neutrophils, the shapeand chromatin morphology of the nucleus may indicate whether it is animmature “band” neutrophil or a mature “segmented” neutrophil. Bandneutrophils are immature neutrophils that have recently emerged from thebone marrow. An increase in the proportion of band neutrophils mayindicate an ongoing infection or inflammation.

The sample was mixed with a fluorescently labeled anti-CD16 antibody,which recognizes CD16, a cell surface receptor on neutrophils. Thesample was also stained with a fluorescent nuclear dye. The sample wasimaged by fluorescence microscopy, to obtain both nuclear staining andCD16 staining data from the cells. Band neutrophils generally havesimilar expression levels of CD16 as mature segmented neutrophils, andthus cannot be distinguished by virtue of fluorescence intensity fromCD16 staining alone.

Image analysis including image segmentation is used to recognize nuclearstaining and morphologies of band neutrophils and segmented neutrophils,thereby allowing classification of the cells. The size, shape, andfluorescence intensity of the nucleus of cells are examined. Inaddition, the nuclei are analyzed to determine the number of lobes(peaks in intensity within the nuclear area), distance between the lobesof the nucleus, and the changes in curvature (second derivative) of thenuclear outline. FIG. 5A shows representative images of bandneutrophils. In these images, the nucleus appears as a light grey, andthe cell cytoplasm appears as a darker grey. As neutrophilsdifferentiate through the myeloid lineage, they develop a characteristic“U” shaped nucleus prior to reaching full maturity. FIG. 5B showsrepresentative images of segmented neutrophils. In these images, thenucleus appears as a light grey, and the cell cytoplasm appears as adarker grey. The nuclei of segmented neutrophils have multiplesegments/lobes (typically about 3-5). Thus, this analysis supportsidentification and quantification of different subpopulations ofneutrophils in the blood. Image segmentation is discussed further inExample 7 below.

Example 5

A sample of cells from a subject with chronic lymphocytic leukemia (CLL)is obtained. The objective is to quantify the extent of CD5 expressionon B-cells from the subject. Anti-CD20 antibodies are selected as thebinder for B-cells. Anti-CD20 antibodies labeled with a first coloredfluorophore are mixed with the sample. After an appropriate incubationtime, the sample is washed and the unbound anti-CD20 antibodies areremoved. The sample is exposed to a light source capable of exciting thefirst fluorophore, and fluorescent signal is measured using aspectrophotometer. Based on the fluorescent signal, the approximateconcentration of B-cells in the sample is determined. The determinedapproximate concentration of B-cells is, in fact, within 1.5 fold of thetrue concentration of B-cells in the sample.

Based on the approximate concentration of B-cells in the sample, anappropriate amount of anti-CD5 binder is added to the sample so that aproportional relationship between CD5 expression and CD5 fluorescence ismaintained. The anti-CD5 binder is coupled to a second fluorophore,which has a different peak excitation wavelength than the firstfluorophore (attached to the anti-CD20 binder). The anti-CD5 antibody isadded to the sample, and then individual cells of the sample are exposedto a light source capable of exciting the second fluorophore, andfluorescent signal from individual cells is measured. Based on thefluorescent signal from cells, the average amount of CD5 in B-cells inthe sample is determined.

Although this example is described in the context of CD5, it should beunderstood that this concept of obtaining an approximate count to guidean addition of a desired amount of material for use in a subsequentstep, is not limited to CD5 and use of this concept with other types ofcells, analytes, or objects is not excluded.

Example 6

Blood cells may be imaged, identified, and quantified according to themethods disclosed herein. For example, two-dimensional images of cellsin a biological sample, where the cells are labeled (e.g., withfluorescent, chemiluminescent, enzymatic, or other labels) and plated(e.g., allowed to settle on a substrate) and imaged by a camera, may beprepared and analyzed as described in the present example. The cameramay include a lens, and may be attached to or used in conjunction with amicroscope. Cells may be identified in the two-dimensional images bytheir attached labels (e.g., from light emitted by the labels).

80 microliters of whole blood obtained from a fingerstick was loadedinto a capped sample container preloaded with 2 mg/ml EDTA. In thisinstance an enclosed sample container was used (with a removable orpierceable cap); it will be understood that any suitable vessel forholding such a small volume sample may be used, including, but notlimited to, a capped vessel or an uncapped vessel. The sample containerwas centrifuged at 1200×g for 5 minutes, to separate the blood cellsfrom the blood plasma. Centrifugation of the sample container resultedin the separation of the blood sample in the sample container into twomajor components (from top of the sample container to the bottom): 1)blood plasma and 2) packed blood cells. This process ensures that nodroplets of blood remain isolated, but coalesce with the main body ofthe liquid. In addition, this process separates the cells from elementsof the plasma thus reducing metabolism and allowing for longer storageof the sample.

The centrifuged sample container was loaded into a cartridge containingmultiple fluidically isolated reagents, tips, and a cytometry cuvette.The cartridge contained all the reagents required for the assay. Thecartridge was loaded into a device equipped with at least a centrifuge,a pipette and a platform to load the cuvette. The pipette in the devicehas a plurality of nozzles, some nozzles being of a different size thansome other nozzles.

Inside the device, a nozzle on the pipette was lowered on a cuvettecarrier tool causing it to engage a corresponding hole on the carriertool. This tool was subsequently moved to the cartridge and lowered onthe cytometer cuvette. Pins on the tool were then able to engagecorresponding holes on the cuvette and pick it up. The cuvette wastransferred to a loading station elsewhere in the device.

Next, inside the device, a larger nozzle of the pipette was lowered intothe cartridge to engage a pipette tip stored in the cartridge. Thepipette and tip together were then used to mix the cells and plasma inthe sample container by positioning the pipette tip within the sample inthe sample container and repeatedly aspirating material into anddispensing material from the tip. Once the cells were resuspended in theplasma so that the whole blood sample was thoroughly mixed, 5microliters of the mixed whole blood was aspirated to provide an aliquotfor measurements of properties of the blood sample. This 5 microliteraliquot was used for measurements directed to the red blood cells andplatelets in the sample. As discussed below, a portion of the sampleremaining after removal of this 5 microliter aliquot was used formeasurements directed at white blood cells in the sample.

The 5 microliters of whole blood was dispensed into a vessel containinga mixture of phosphate buffered saline and 2% by weight of bovine serumalbumin, to dilute the whole blood twenty-fold (resulting in 100microliters of diluted sample). After mixing vigorously, 5 microlitersof this sample was transferred to another vessel containing a cocktailof labeling antibody reagents: anti-CD235a conjugated to Alexa Fluor®647 (AF647), anti-CD41 and anti-CD61 conjugated to phycoerythrin (PE).The mixture was incubated for 5 minutes. Subsequently, 10 microliters ofthis mixture was mixed with 90 microliters of a buffer containing azwitterionic surfactant at <0.1% by weight. The surfactant moleculesmodify bending properties of the red cell membrane such that all cellsassume a stable spherical shape. This transformation is isovolumetric asthe buffer used is isotonic with cytoplasm; thus no osmotically drivenexchange of fluid can occur across the cell membrane. After incubatingthis for another 2 minutes, 30 microliters of this solution was mixedwith a solution containing glutaraldehyde, a fixative andnon-fluorescent beads of 10 micron (μm) diameter. The mixture had afinal concentration of 0.1% glutaraldehyde and 1000 beads permicroliter. Glutaraldehyde rapidly fixes cells thus preventing celllysis and other active biological processes.

In this non-limiting example, the pipette then engaged a tip in thecartridge, aspirated 7 μL of the above mixture of and loaded the 7 μLinto a channel within the cuvette placed on a platform with the carriertool. After the mixture was loaded in into cuvette, the pipetteaspirated 10 μL of mineral oil from a vessel in the cartridge, andplaced a drop of mineral oil on both open ends of the loaded channel ofthe cuvette. Hexadecane was added to the ends of the open channel toprevent evaporation of liquid from the loaded cuvette channel (mineraloil would also work). Next, the device-level sample handling apparatusengaged the cuvette carrier/cuvette combination, and transported thecuvette carrier/cuvette combination from the module containing thecartridge to the cytometry module of the device. At the cytometrymodule, the device-level sample handling apparatus placed the cuvettecarrier/cuvette combination on the microscopy stage of the cytometrymodule. The time required for these operations, in addition to a 2minute wait time allowed the swelled cells to settle to the floor of thecuvette prior to imaging.

After the cuvette carrier/cuvette was placed on the microscopy stage,the stage was moved to pre-determined location so that the opticalsystem of the cytometer could view one end of the channel containing thesample. At this location, the optical system relayed images of thesample acquired with dark field illumination from a ringlight. Theseimages coupled with actuation of the optical system on an axisperpendicular to the plane of the cuvette were used to find the plane ofbest focus. Once focused, the optical system was used to acquirefluorescence images of the sample at different wavelengths, commensuratewith the fluorophores that were being used. For example, to visualizered blood cells that had been labeled with anti-CD235 conjugated toAlexa Fluor® 647, a red (630 nm wavelength) light source was used toexcite the sample and wavelengths between 650 nm and 700 nm were used toimage the sample. A combination of a polychroic mirror and a bandpassemission filter was used to filter out unwanted wavelengths from theoptical signal. Since the cells had settled on the floor of the cuvette,images at a single plane of focus were sufficient to visualize all cellsin the region.

Data from the images was processed by a controller associated with thesample processing device. The image processing algorithms employed hereutilized fluorescence images of cells to detect them using a combinationof adaptive thresholding and edge detection. Based on local intensityand intensity gradients, regions of interest (RoI) were created aroundeach cell. Using dark field images, beads in the sample were alsoidentified and RoIs were created around the beads. All the RoIs in eachfield of view were enumerated and their intensity in each image of thatfield of view were calculated. The information output by the imageprocessing algorithm consisted of shape or morphometric measurements andfluorescence and dark field intensities for each RoI. This informationwas analyzed using statistical methods to classify each object as eithera red blood cell (positive for CD235a, but negative for CD41/CD61), aplatelet (positive for CD41/CD61 and negative CD235a) or a bead. Theshape descriptors such as perimeter, diameter and circularity were usedto calculate the volume of each red blood cell and platelet. Since thebeads were added at a known concentration, the average ratio of beads tocells over the whole channel was used to calculate cell concentration interms of cells/microliter. Based on the steps performed for processingthe sample, this concentration was corrected for dilution to arrive atconcentration of cells in the original whole blood sample. The followingquantities were calculated from a sample: 1) number of red blood cellsin the cuvette; 2) average volume of red blood cells in the cuvette; 3)red blood cell distribution width (RDW) of red blood cells in thecuvette; 4) number of platelets in the cuvette; and 5) average volume ofplatelets in the cuvette. Based on these calculations, the following wascalculated for the original blood sample.

Exemplary Measured Value Result Range Concentration of red blood cells4.8 4-6 (million cells per microliter) Mean volume of red blood cells,femtoliter 88  80-100 red blood cell distribution width (RDW), (%) 12  11-14.6 Concentration of platelets 254 150-400 (thousand cells permicroliter) Mean volume of platelets, femtoliter 10.4  7.5-11.5

After removal of the 5 microliter aliquot used for analysis of RBC andplatelet information, the remaining 75 microliters of sample was used toanalyze the white blood cell population of the whole blood sample. Theremaining 75 microliters of whole blood had also been mixed byrepeatedly aspirating and dispensing the sample within the same thevessel by the pipette. Approximately 40 microliters of the remaining 75microliters of mixed whole blood was aspirated into a pipette tip, andtransferred by the pipette to a centrifuge tube in the cartridge. Thecentrifuge tube containing the blood sample was engaged by the pipette,and transferred to and deposited in a swinging bucket in a centrifugewithin the module. The centrifuge was spun to provide 1200×g for 3minutes, separating the blood into EDTA-containing plasma as thesupernatant and packed cells in the pellet.

After centrifugation, the centrifuge tube was removed from thecentrifuge and returned to the cartridge. The plasma supernatant wasremoved by the pipette and transferred to a separate reaction vessel inthe cartridge. From a reagent vessel in the cartridge, 16 microliters ofresuspension buffer was aspirated by the pipette, and added to the cellpellet in the centrifuge tube. The pipette then resuspended the cellpellet in the resuspension buffer by repeatedly aspirating anddispensing the mixture in the centrifuge tube. Next, the pipetteaspirated 21 microliters of the resuspended whole blood and added it toanother vessel containing 2 microliters of anti CD14-Pacific Blue™ andDRAQ5®, mixed, and incubated for 2 minutes. Twenty microliters of thismixture was then added to 80 microliters of a lysis buffer. The lysisbuffer was a solution including saponin (a gentle surfactant; othersurfactants which may be used include anionic, cationic, zwitterionic,and non-ionic surfactant compounds, e.g., as discussed above) andparaformaldehyde (a fixative; other fixatives which may be used includeformaldehyde, glutaraldehyde, and other cross-linking agents). Thedetergent causes a large number of holes to be formed in the membranesof cells. Red blood cells, due to their unique membrane properties, areparticularly susceptible to this hole formation and lyse completely,their contents leaking out into the liquid around. The presence of thefixative prevents unintentional lysis of the white blood cells.Platelets also remain unlysed. The purpose of this step is to removeintact red blood cells from the mixture as they outnumber white bloodcells by about 1000:1. Platelets do not interfere with imaging and henceare irrelevant to this process. The lysis buffer also contained 10 μMnon-fluorescent beads at a known concentration.

After a 5 minute incubation, the vessel was spun again at 1200×g for 3minutes. The supernatant was aspirated by a pipette tip, removing thered blood cell ghosts and other debris, and deposited into a waste areain the cartridge. Approximately 15 microliters of liquid with packedwhite blood cells were present in the cell pellet.

In order to determine a rough approximation of the number of white bloodcells present in the cell pellet, the pipette first resuspended thewhite blood cells in the vessel and then aspirated the liquid fortransport to and inspection by a spectrophotometer. The white blood cellsuspension was illuminated with light at a wavelength of 632 nm, whichis the excitation wavelength for Alexa Fluor® 647 dye and DRAQ5®. Thelight emitted by the cell suspension was filtered by a 650 nm long passfilter and measured in the spectrophotometer. This measurement wascorrelated with previously generated calibration curve to estimate arough concentration of white blood cells in the cell suspension.Typically, cell concentrations ranged from about 1000 cells permicroliter to about 100,000 cells per microliter. This estimate was usedto calculate an appropriate dilution factor to ensure that theconcentration of cells in the cuvette was constrained to within atwo-fold range around a pre-defined target concentration. The purpose ofthis step was to ensure that cells are not present at too high or toolow a density on the cuvette. If the cell density is too high, theaccuracy of image processing algorithms is compromised, and if the celldensity is too low, an insufficient number of cells are sampled.

Based on the dilution factor calculated in the above step, a diluentcontaining labeled antibodies against CD45 (pan-leukocyte marker), CD16(neutrophil marker) and CD123 (basophil marker) was added to the cellsuspension and mixed.

Once the cuvette in complex with cuvette carrier was placed on thecuvette carrier block, 10 microliters of the mixture of white bloodcells resuspended in cytometry buffer was loaded into each of twochannels in the cuvette. After the mixture was loaded into channels ofthe cuvette, the pipette aspirated 10 μl of hexadecane from a vessel inthe cartridge, and placed a drop of mineral oil on both open ends ofboth channels in the cuvette loaded with white blood cells.

Next, the device-level sample handling apparatus engaged the cuvettecarrier/cuvette combination, and transported the cuvette carrier/cuvettecombination from the module containing the cartridge to the cytometrymodule of the device. At the cytometry module, the device-level samplehandling apparatus placed the cuvette carrier/cuvette combination on themicroscopy stage of the cytometry module. After the cuvettecarrier/cuvette was placed on the microscopy stage, the two channels ofthe cuvette containing white blood cells were imaged as described abovefor the RBC/platelet mixture.

Dark field images of the white blood cells were used to count thenumbers of cells in a field (as shown in FIG. 9A). Cell surface markerswere used to determine the cell type of individual white blood cells inan image; for example, CD14 marks monocytes; CD123 marks basophils; CD16marks neutrophils; and CD45-AF647 were used to mark all leukocytes(FIGS. 9B-9E). The nuclear stain DRAQ5® was used to mark cell nuclei,and so to differentiate nucleated cells (such as white blood cells) frommature red blood cells, which have no nucleus (FIG. 9F).

The image processing algorithms employed here utilized fluorescenceimages of cells to detect them using a combination of adaptivethresholding and edge detection. Based on local intensity and intensitygradients, boundaries of regions of interest (RoI) were created aroundeach cell. Using dark field images, beads in the sample were alsoidentified and RoI boundaries were created around the beads. All theRoIs in each field of view were enumerated and their intensity in eachimage of that field of view were calculated. The information output bythe image processing algorithm consisted of shape or morphometricmeasurements and fluorescence and dark field intensities for each RoI.This information was analyzed using statistical methods to classify eachobject as a lymphocyte, monocyte, basophil, eosinophil, neutrophil or abead. Based on enumeration of cells of different types, thecorresponding bead count and the dilution ratio implemented duringsample processing, an absolute concentration of cells per microliter oforiginal whole blood was calculated. This was calculated for all whiteblood cells and each subtype, and reported as both absoluteconcentration (cells per microliter) and proportion (%).

Examples of images and plots of results of such measurements arepresented in FIGS. 9A-9F, 10, and 11A-11D.

FIGS. 9A-9F show representative images of blood cells from a sample ofwhole blood; these images were taken using different imaging techniquesand dyes. The image shown in FIG. 9A was taken of cells from whole bloodusing dark-field illumination. The image shown in FIG. 9B was taken ofcells from whole blood showing fluorescence from anti-CD14 antibodieslabeled with Pacific Blue dye; the fluorescent cells are monocytes. Theimage shown in FIG. 9C was taken of cells from whole blood showingfluorescence from anti-CD123 antibodies labeled with PECy5 dye; thefluorescent cells are basophils. The image shown in FIG. 9D was taken ofcells from whole blood showing fluorescence from anti-CD16 antibodieslabeled with PE dye; the fluorescent cells are neutrophils. The imageshown in FIG. 9E was taken of cells from whole blood showingfluorescence from anti-CD45 antibodies labeled with AF647 dye; allleukocytes fluoresce under these conditions. The image shown in FIG. 9Fwas taken of cells from whole blood dyed with DRAQ5® to stain cellnuclei. Thus, leukocytes and platelets are stained and fluoresce underthese conditions, but red blood cells (lacking nuclei) are not stainedand do not fluoresce.

FIG. 10 shows a representative composite image of cell-types in wholeblood from images acquired according to the methods disclosed herein.Images of a monocyte (labeled and seen in the upper left quadrant of thefigure, with a reddish center surrounded by a blue-purple ring), alymphocyte (labeled and seen in the center of the figure, with a brightred center surrounded by a dimmer red ring), an eosinophil (labeled andseen in the lower left quadrant of the figure, with a green centersurrounded by a red border), and a neutrophil (labeled and seen in thelower right quadrant of the figure, with a green center surrounded by ayellow and green border) are shown in the figure.

It is of interest to identify and quantify various cell types found insuch blood samples. There may be multiple ways to approach such aclassification process, which, in some embodiments, may be considered asbeing a statistical problem for multi-dimensional classification. Itwill be understood that a wide variety of methods are available in thefield to solve these types of classification problems. A particularembodiment of such an analysis is provided below.

FIGS. 11A-11D show plots of various cell types identified and quantifiedby the cytometric assays described in this example. FIG. 11A shows aplot of spots (cells) by intensity of the marker FL-17 (anti-CD14antibody labeled with pacific blue dye) versus intensity of FL-9 (darkfield scatter signal) to identify monocytes. FIG. 11B shows a plot ofspots (cells) by intensity of the marker FL-19 (anti-CD123 antibodylabeled with PE-CY5 dye) versus intensity of the marker FL-15 (anti-CD16labeled with PE dye) to identify basophils. FIG. 11C shows a plot ofspots (cells) by intensity of the marker FL-15 (anti-CD16 labeled withPE dye) versus intensity of the marker FL-11 (anti-CD45 antibody labeledwith AF647 dye) to identify lymphocytes. FIG. 11D shows a plot of spots(cells) by intensity of the marker FL-15 (anti-CD16 labeled with PE dye)versus intensity of FL-9 (dark field scatter signal) to identifyneutrophils and eosinophils.

The initial identification of monocytes (9.6%, as shown in FIG. 11A) isused to guide the subsequent identification of basophils (0.68%, asshown in FIG. 11B). The identification of monocytes and basophils asshown in FIGS. 11A and 11B is used to guide the subsequentidentification of neutrophils and eosinophils (68% neutrophils, 3.2%eosinophils, of the WBCs shown in FIG. 11D). Finally, lymphocytes areidentified as shown in FIG. 11C (93% of the WBCs plotted in FIG. 11C,corresponding to 18% of the cells in the original sample).

The present methods correlate well with other methods. Counts of whiteblood cells, red blood cells, and platelets were made with samples ofEDTA-anti coagulated whole blood. The white blood cells were furthercounted to determine the numbers of neutrophils, monocytes, andlymphocytes in the sample. In the measurements shown in FIG. 12,EDTA-anti coagulated whole blood samples were split into two, and onepart of the samples were run on the system disclosed herein, using themethods disclosed herein. The other part of the samples was run on anAbbott CELL-DYN Ruby System (Abbott Diagnostics, Lake Forest, Ill.,USA), a commercial multi-parameter automated hematology analyzer. Acomparison of the results obtained with both methods is shown in FIGS.12A-12F.

As shown in FIGS. 12A-12C, the numbers of white blood cells (“WBCs”,FIG. 12A), red blood cells (“RBCs”, FIG. 12B) and platelets (FIG. 12C)measured by the present methods correlate well with the numbers of WBCs,RBCs, and platelets measured by other methods in corresponding aliquotsof the same samples as were analyzed by the present methods. As shown inFIGS. 12D-12F, the numbers of neutrophils, monocytes, and lymphocytesmeasured by either method were very similar, and correlated well witheach other. In embodiments of the methods disclosed herein, bloodsamples may be diluted to reduce or eliminate red blood cell overlap.For example, samples in which red blood cell counts were obtained weretypically diluted by about 400-fold to about 1000-fold so that the redblood cells would be sufficiently separated for accurate counting. Whereadvantageous or required, such dilutions were performed by sequentialdilution (e.g., where a sample or portion thereof was diluted a firsttime to provide a first diluted sample, and that first diluted sample(or portion thereof) was further diluted one, two, or more times, asneeded to provide the desired dilution). As discussed above, beads maybe incorporated into such diluted samples to provide an independentmeasure of the dilution: since the number of beads added is known, acount of the number or concentration of beads in the final (diluted)sample may be used to calculate the actual amount of dilution that wasobtained. Typically, a ratio of about 5-7 RBCs per bead provides adesirable ratio of RBCs to beads. Optionally, the solution may also havea component that prevents the beads for adhering to each other. In onenon-limiting example, the use of a known number or concentration ofreference bodies such as beads or other structures can be particularlyuseful when they are added to undiluted sample prior to dilution step,especially in serial dilution steps used to create 200-fold or higherdilutions. As long as the sample and beads are well mixed before eachaspiration step, this reduces the impact of inaccuracies in dilutionsteps and makes the method insensitive to dispense errors in thesemultiple dilution steps. Optionally, some embodiment may add thereference bodies after the first dilution step of a multiple stepdilution process. Optionally, some embodiment may add the referencebodies after the second dilution step of a multiple step dilutionprocess.

In aspects of the term as used herein, the term “cytometry” refers toobservations, analysis, methods, and results regarding cells of abiological sample, where the cells are substantially at rest in a fluidor on a substrate. Cells detected and analyzed by cytometry may bedetected and measured by any optical, electrical or acoustic detector.Cytometry may include preparing and analyzing images of cells in or froma biological sample (e.g., two-dimensional images). The cells may belabeled (e.g., with fluorescent, chemiluminescent, enzymatic, or otherlabels) and plated (e.g., allowed to settle on a substrate) and,typically, imaged by a camera. A microscope may be used for cell imagingin cytometry; for example, cells may be imaged by a camera and amicroscope, e.g., by a camera forming an image using a microscope. Animage formed by, and used for, cytometry typically includes more thanone cell.

Example 7

This example presents a method and results of sequential segmentation ofwhite blood cell images from samples of blood. Other suitable methodsinclude summing images, including providing weighted averages ofmultiple images, to provide images for use in determining cellboundaries. Nuclear staining dyes and other dyes may be used, includinglabeled antibodies for binding to specific cell markers, either togetheror separately, to obtain images for analysis. For example, cell size andcell boundary estimates may be obtained using images obtained with eachdye or marker separately, or some or all images may be combined foranalysis. The present methods provide related and improved methods forestimating cell size and for determining boundaries of cells imaged bydevices and systems as disclosed herein. It will be understood thatthese methods are useful for the analysis of cells imaged by otherdevices and systems as well.

Segmentation is useful for determining contours of images, e.g., fordetermining contours (e.g., boundaries, such as optimal outlines) ofimages of objects within a larger image containing one or more objectsand (typically) background or other features as well. Sequentialsegmentation is an iterative process which, when applied to images ofcells in a biological sample, may be used to provide progressivelybetter cell contours by use of successive procedures which result in afinal, optimal (or sufficiently accurate) result. The results presentedin the present example demonstrate the use of sequential segmentation offluorescence images of individual white blood cells using nuclear stainsto provide regions of interest within the cells (e.g., to provide imagesof cell nuclei) which are used as the seed upon which to base thesequential segmentation process for determining the outer boundaries ofthe cells containing those nuclei.

Dyes and stains useful for such images and the analysis thereof includethe dyes and markers disclosed herein, such as, e.g., DAPI, DRAQ5®,propidium iodide, or other DNA-staining dye; PE, Pacific Blue™,allophycocyanin (APC), Alexza Fluor®, and other dyes.

Segmentation was applied to WBC images obtained using fluorescencemicroscopy images to find contours (e.g., optimal cell outlines) foreach cell that separated them from the image background. A cell regionof interest (ROI) was defined as the region interior to the contour, andwas used to compute shape and size metrics such as area, volume, andcircularity, as well as intensity measures such as mean, median, minimumand maximum intensity. In FIG. 15B, examples of cells (bright),background (dark), and contours (red) are shown. The contours shown inFIG. 15B were determined by sequential segmentation as described in thisexample.

For each field of view (FoV), multiple fluorescence images were acquiredwith different filters, each emphasizing different WBC types. Examplesof different fluorescent image types can be seen in FIG. 13A-E); FIG.13A is a dark-field image, FIG. 13B is from labeled anti-CD45antibodies, FIG. 13C imaged with nuclear stain DRAQ5®, FIG. 13D is fromlabeled anti-CD16 antibodies, and FIG. 13E is from labeled anti-CD123antibodies.

FIGS. 13A-13E show white blood cell (WBC) images obtained usingmicroscopy, for use in performing sequential segmentation analysis todetermine contours for each cell and to thus differentiate the cellimages from the background images. FIG. 13A is a dark field image; FIG.13B is a fluorescence image showing cell labelling by anti-CD45antibodies; FIG. 13C is a fluorescence image cells labelling by thenuclear stain DRAQ5®; FIG. 13D is a fluorescence image showing celllabelling by anti-CD16 antibodies; and FIG. 13E is a fluorescence imageshowing cell labelling by anti-CD123 antibodies.

The assumption of the segmentation method was that the desired cellcontour for a given nucleus could be found in the image in which thecell area was the largest. The method consisted of the following steps:

-   -   1) Segmentation of cell nuclei using the image stained with        DRAQ5®.    -   2) For each acquired image: grow cell regions using watershed        segmentation, initialized with the segmented cell nuclei.    -   3) For each nucleus: find the cell ROI with the largest area        across all images and register that as the final segmentation        for that cell.

Cell nuclei were detected using the image stained with DRAQ5®. ROIs werefound using adaptive thresholding, where a pixel's intensity was set asforeground if its intensity was a certain amount higher than the meanintensity in the pixel neighborhood. Pixel intensity varied acrossimages; for example, pixel intensity decreased with distance from localmaximal intensity values. The rate of change in such decrease of pixelintensity (with increasing distance from local maxima) was used todetermine boundaries, or edges, of the imaged objects. Sizes of imagedobjects (e.g., cell nuclei when DRAQ® or other nuclear stain was used)were then calculated using the boundaries. An ROI was classified as anucleus if it was within an allowable size range. FIG. 14C shows animage stained with DRAQ5®, showing nuclei contours identified in thisway in blue.

Each nucleus ROI was assumed to be in the interior of a cell ROI. Cellsegmentation in an image was performed by growing the regions around thealready segmented nucleus ROIs. The stopping criteria can be based ongradient magnitude, intensity information, or other factors or acombination of factors. Examples of segmentation techniques that can beused are active contours, geodesic active contours, and watershed. Thewatershed algorithm was used, and the ROI growing was stopped eitherwhen it reached a maximum in the intensity gradient magnitude, asignificant intensity decrease, or when it encountered a neighboringROI.

The watershed segmentation was performed on each image acquired for theFoV, and the cell ROIs were stored. For each nucleus in the image thecell ROI areas were compared across all images, and the ROI with thelargest area was recorded as the final cell ROI for that nucleus. Allcell ROIs with maximum area were then combined into a final WBCsegmentation. An example of a final sequential WBC segmentation is shownin FIG. 15B. This method determines cell regions more accurately than doother methods, e.g., more accurately finds cell regions than does aone-pass segmentation of a weighted average of fluorescence images. Thecontours shown in FIG. 15A were determined by watershed segmentationperformed once on the composite cell images, while the contours shown inFIG. 15B were determined by sequential WBC segmentation as describedherein.

FIGS. 14A-14E show the images in FIGS. 13A-13E with segmentationresults. Nucleus ROIs are plotted using blue contours and cell ROIs havered contours. FIG. 14A is a dark-field image with nuclei ROIs overlaidin blue and the generated cell segmentation in red, FIG. 14B is fromlabeled anti-CD45 antibodies with nuclei ROIs overlaid in blue and theresulting cell segmentation in red, FIG. 14C imaged with nuclear stainDRAQ5® with segmented nuclei ROIs in blue, FIG. 14D is from labeledanti-CD16 antibodies with nuclei ROIs in blue and the resulting cellsegmentation in red, and FIG. 14E is from labeled anti-CD123 antibodieswith nuclei ROIs overlaid in blue and the resulting cell segmentation inred.

FIGS. 14A-14E show white blood cells (WBCs) images obtained usingmicroscopy, as in FIGS. 13A-13E, for performing sequential segmentationanalysis to determine external (e.g., cell membrane) and internal (e.g.,nucleus) contours for each cell and to thus identify the cell nucleus aswell as to differentiate the cell ROIs from the background regions. Thelines within the cell images identify the boundaries of the WBC nucleusfor each cell as determined by sequential segmentation analysis. FIG.14A is a dark field image; FIG. 14B is a fluorescence image showing celllabelling by anti-CD45 antibodies; FIG. 14C is a fluorescence imagecells labelling by the nuclear stain DRAQ5®; FIG. 14D is a fluorescenceimage showing cell labelling by anti-CD16 antibodies; and FIG. 14E is afluorescence image showing cell labelling by anti-CD123 antibodies.

Another approach to WBC segmentation was to perform a weighted averageof all the fluorescent and the dark-field image and perform watershedsegmentation once on that composite image. This method may create a biastowards cells that had more staining across the images. FIG. 15A shows acomposite image. ROIs from watershed segmentation performed once on thecomposite image are show in red contours. FIG. 15B shows a compositeimage with the described sequential WBC segmentation plotted with redcontours. The main contributors to the final segmentation were from FIG.14B and FIG. 14D in this case.

FIGS. 15A-15B show composite images of white blood cells (WBCs) shown inFIGS. 13A-13E and 14A-14E. FIG. 15A is a composite image of the cellsshown in FIGS. 13A-13E and 14A-14E, with cell contours obtained bywatershed segmentation performed once. FIG. 15B is a the result ofsequential segmentation as described herein applied to the compositeimage of the cells shown in FIGS. 13A-13E and 14A-14E, showing cellcontours obtained by that analysis. The sequential segmentation analysisillustrated in FIG. 15B appears to better identify cell contours thandoes the watershed segmentation performed once as shown in FIG. 15A.

Optical Systems

Referring now to FIGS. 6A and 6B, embodiments of an optical systemsuitable for use herein will now be described. Although theseembodiments of the system are described in the context of being able toperform cytometry, it should also be understood that embodiments of thesystem may also have uses and capabilities beyond cytometry. By way ofexample and not limitation, the imaging and image processingcapabilities of the systems disclosed herein may be used for manyapplications, including applications outside of cytometry. Since imagesof the sample being analyzed are captured, and image information istypically linked or associated in the system to quantitativemeasurements, one can further analyze the images associated with thequantitative information to gather clinical information in the imagesthat would otherwise be unreported.

A sample to be analyzed, e.g., by cytometry or other optical or imagingmeans, may be held in a sample holder for analysis. For example, acuvette may serve as such a sample holder. The embodiment shown in FIG.6A shows a perspective view of a cuvette 600 that has a plurality ofopenings 602 for receiving a sample or portion thereof for analysis. Forexample, an opening 602 may be used as an entry port to provide asample, such as a fluid sample, to a channel, conduit, or chamber (e.g.,a sample chamber) for analysis. The horizontal cross-sectional shape ofthe embodiment of FIG. 6A is a rectangular horizontal cross-sectionalshape. Although the system is described in the context of a cuvette, itshould be understood that other sample holding devices may also be usedin place of or in combination with the cuvette 600.

As seen in the embodiment of FIG. 6A, the openings 602 may allow for asample handling system (not shown) or other delivery system to depositsample into the opening 602 which may be connected with, and may leadto, an analysis area 608 in the cuvette where the sample can beanalyzed. In one non-limiting example, an analysis area 608 may be achamber. In another non-limiting example, an analysis area 608 may be achannel. In embodiments, an analysis area 608 that is configured as achannel may connect two entry ports 602. In a still further non-limitingexample, an analysis area 608 may be a channel wherein the sample isheld in a non-flowing manner. In any of the embodiments herein, thesystem can hold the samples in a non-flowing manner during analysis.Optionally, some alternative embodiments may be configured to enablesample flow through the analysis area before, during, or after analysis.In some embodiments, after analysis, the sample is extracted from thecuvette 600 and then delivered to another station (in a system havingmultiple stations) for further processing or for further processing oranalysis. Some embodiments may use gate(s) in the system to controlsample flow.

FIG. 6A shows that, in some embodiments of a cuvette 600, a cuvette 600may have a plurality of openings 602. Sample may be added to the sampleholder via entry ports 602. An opening 602 may be operably connectedwith (e.g., in fluid continuity with) an analysis area 608. An analysisarea 608 may be operably connected with (e.g., in fluid continuity with)a plurality of openings 602. It will be understood that some embodimentsmay have more, or may have fewer, openings 602 in the cuvette 600. Someembodiments may link certain openings 602 such that selected pairs orother sets of openings 602 can access the same channel (e.g., analysisarea 608 that is configured as a channel). By way of non-limitingexample, there may an opening 602 at each end of an analysis area 608.Optionally, more than one opening 602 may be at one end of an analysisarea 608.

Embodiments of a cuvette 600 may have structures 610 that allow for asample handling system to engage and transport the cuvette 600. Acuvette 600 as illustrated in FIG. 6A and FIG. 6B may be engaged by asample handling system via an element 610, effective that the cuvette600 may be transported from one location to another. An element 610 mayalso be used to secure a cuvette 600 at a desired location, e.g., priorto, or following transport to a location (such as over a detector foroptical imaging and analysis), a cuvette 600 may be held in position byan element 610 or by a tool or device which uses an element 610 to holda cuvette 60 in position. In one non-limiting example, the structures610 can be openings in the cuvette 600 that allow for a pipette or otherelongate member to engage the cuvette 600 and transport it to thedesired location. Optionally, in place of or in combination with saidopening(s), the structures 610 can be, or may include, a protrusion, ahook, a magnet, a magnetizable element, a metal element, or otherfeature that can be used to engage a cuvette transport device. Inembodiments, force (e.g., compression, or other force) may be applied toa cuvette 600; for example, compression may be applied to a cuvette 600in order to press a cuvette 600 onto a substrate or surface (e.g., asurface of a base support 620), effective to place the cuvette 600 ineffective optical contact with the surface. In embodiments, such force(e.g., compression) may aid in providing desired optical properties,such as providing good contact between a cuvette 600 and a base support620, effective to allow passage of light without significant distortionat the interface, or without significant reflection at the interface, orother desired optical property. In embodiments, such force (e.g.,compression) may be applied, at least in part, via a structure 610 orvia multiple structures 610.

As shown in FIG. 6B (in perspective view), a cuvette 600 may have acircular horizontal cross-sectional shape. An opening 602 (or multipleopenings 602, which may be present in similar embodiments, not shown inthe figure) may allow sample handling system or other delivery system todeposit sample into the opening 602 which may then lead to an analysisarea 608 in the cuvette where the sample can be analyzed. Non-limitingexamples of suitable analysis areas 608 include an analysis area 608comprising a chamber, and an analysis area comprising a channel. Inembodiments, such an analysis area 608 may be located within an annularstructure such as the annular structure 604 shown in FIG. 6B. Inembodiments, an opening 602 may be connected with an analysis area 608.In embodiments, an analysis area 608 within a structure 604 may form acontinuous ring-shaped chamber, connecting with an opening 608 effectiveto allow flow within the chamber in either of two directions away froman opening 602. In embodiments, an analysis area 608 within a structure604 may form a ring-shaped channel or chamber, with one end connectingwith an opening 608, and another end separated or blocked off from theopening 602, effective to allow flow within the chamber in only onedirection away from an opening 602. In embodiments, such a one-wayring-shaped channel or chamber may have a vent or other aperture at alocation distal to an opening 602. In a still further non-limitingexample, the analysis area may be or include a channel wherein thesample is held in a non-flowing manner; a sample may be held in anon-flowing manner in an analysis area 608 that comprises a ring-shapedchannel, whether the ring-shaped channel is connected to an opening 602from two directions, or whether the ring-shaped channel is connected toan opening 602 from only a single direction. In any of the embodimentsherein, the system can hold the samples in a non-flowing manner duringanalysis. Optionally, some alternative embodiments may be configured toenable sample flow through the analysis area before, during, or afteranalysis. In some embodiments, after analysis, the sample is extractedfrom the cuvette 600 and then delivered to another station (in a systemhaving multiple stations) for further processing or analysis. Someembodiments may use gate(s) in the system to control sample flow.

FIG. 6B shows only a single annular structure 604; however, it will beunderstood that, in further embodiments of a cuvette 600 shaped asillustrated in FIG. 6B, a cuvette 600 may have a plurality of annularstructures 604. For example, a cuvette 600 having a plurality of annularstructures 604 may have concentric annular structures 604, of differentsizes, with an outer annular structure 604 surrounding one or more innerannular structures 604. Such annular structures 604 may include analysisareas 608 within each annular structure 604. FIG. 6B shows only a singleopening 602; however, it will be understood that, in further embodimentsof a cuvette 600 shaped as illustrated in FIG. 6B, a cuvette 600 mayhave a plurality of openings 602. For example, a cuvette 600 having aplurality of annular structures 604 (e.g., having a plurality ofconcentric annular structures 604) may have a plurality of openings 602(e.g., each annular structure 604 may have at least one opening 602). Itwill be understood that some embodiments may have more, or may havefewer, openings 602 in a cuvette 600. Some embodiments may link certainopenings 602 such that selected pairs or other sets of openings 602 canaccess the same channel or chamber. By way of non-limiting example,there may an opening 602 at each end of an analysis area. Optionally,more than one opening 602 may be at one end of an analysis area 608.

Some embodiments of cuvettes as illustrated in FIGS. 6A and 6B mayprovide structures 604 over select areas of a cuvette 600. In oneembodiment, the structures 604 are ribs that provide structural supportfor areas of the cuvette that are selected to have a controlledthickness (e.g., areas 613). For example, the thickness may be selectedto provide desired optical properties, including desired pathways forlight to follow before and after reflection within the cuvette 600. Suchreflection may be partial internal reflection (PIR) or total internalreflection (TIR). Whether such reflection occurs depends on manyfactors, including the light wavelength; the angle of incidence of thelight reaching a surface; the composition of the material (of area 613and of an environment or material outside the boundary of an area 613);and other factors. In the embodiments shown in FIG. 6A, the structures604 are rectangular in shape, and have a rectangular cross-section. Inthe embodiments shown in FIG. 6B, the structures 604 are annular inshape, and may have a rectangular cross-section, or a trapezoidalcross-section, or other shaped cross-section. Such structures may haveany suitable cross-sectional shape. As illustrated in FIG. 8B, suchstructures 604 may have a triangular cross-section (e.g., forming asaw-tooth shaped cross-section when multiple ribs are present). It willbe understood that such structures 604 may have other shapes andcross-sections as well (e.g., semi-circular, elliptical, irregular, orother shape), and that, in embodiments, more than one shape may bepresent in the same system (e.g., a cuvette may include rectangular,triangular, or other shaped structures). The structures 604 may be usedwhen the controlled thickness areas 613 are at a reduced thicknessrelative to certain areas of the cuvette and thus could benefit frommechanical support provided by structures 604.

In addition to providing structural support, structures 604 may beuseful to provide material and pathways for internal reflection of lightwithin a cuvette 600. As shown in FIGS. 8A-8D, light reflected within acuvette 600 may include pathways for light reflected within a structure604 (e.g., a rib, or a structure having a triangular cross-section, asshown in the figures, or any other shape, such as a circular orsemi-circular cross-section, or other cross-sectional shape). Structures604 may thus provide convex features extending outwardly from a surface614 of a cuvette 600; or may provide concave features extending inwardlyfrom a surface 614 of a cuvette 600; or may provide both concave andconvex features on a surface 614 of a cuvette 600. Thus structures 604thus may provide mechanical support to a cuvette 600, may providedesired optical properties, including optical pathways, to a cuvette600, and may provide other desirable and useful features andcapabilities to a cuvette 600 as disclosed herein.

Support structures 604 thus may be useful to provide structural support,including, e.g., stiffness, to a cuvette 600. The optical properties ofa cuvette 600 may be important to their use in optical imaging and otheroptical measurements of samples in an analysis area 608 and of cells,particles, and other components of such samples. Maintenance of theproper flatness of a surface of a cuvette 600, including maintenance ofthe flatness of a base portion 606, or a surface 614 or 618; maintenanceof proper orientation and configuration of a cuvette 600 (e.g., withouttwisting, flexing, or other distortion); and maintenance of properpositioning of a cuvette 600 (e.g., on a base support 620, or within anoptical set-up) may be important to the integrity of opticalmeasurements and images obtained using the cuvette 600. Thus, forexample, the design and construction of support structures 604 and baseportion 606 may be important factors in providing and maintaining theproper optical properties of a cuvette 600. Maintenance of the properdimensions of an analysis area 608, including maintenance of the properdistances and relative angles of upper and lower surfaces (or of sidewalls) of an analysis area 608 may be important to providing correct andconsistent illumination of a sample within an analysis area 608.Maintenance of the proper dimensions of an analysis area 608 may also beimportant to insuring that the volume of an analysis area 608, and sothe volume of sample within the analysis area 608, is correct. Asdiscussed herein, force (e.g., compression) may be applied to a cuvette600 to further insure proper flatness, or to decrease twisting ordistortion, or otherwise to insure proper shape, size, and orientationof a cuvette during use. It will be understood that compression may notbe required to insure such proper flatness and proper shape, size, andorientation of a cuvette during use. For example, in embodiments,structures 604 alone may be sufficient to aid or insure that a cuvette600 has the proper flatness and proper shape, size, and orientationduring use. In addition, it will be understood that, in embodiments,compression alone may be sufficient to aid or insure such properflatness and proper shape, size, and orientation of a cuvette 600 duringuse. It will be understood that, in embodiments, the combination ofstructures 604 and compression may aid or insure the maintenance ofproper flatness and proper shape, size, and orientation of a cuvetteduring use.

A cuvette 600, including a support structure 606 and cover portion 612,may be made of any material having suitable optical properties. Inembodiments, a cuvette 600, including a support structure 606 and coverportion 612, may be made of glass (e.g., quartz, or borosilicate glass,or aluminosilicate glass, or sodium silicate glass, or other glass). Inembodiments, a cover portion 612 or a base support 620 may be made of anacrylic, or a clear polymer (e.g., a cyclo-olefin, a polycarbonate, apolystyrene, a polyethylene, a polyurethane, a polyvinyl chloride, orother polymer or co-polymer), or other transparent material. In additionto the optical properties of such materials, the physical properties(e.g., hardness, stiffness, melting point, ability to be machined, andother properties), compatibility with other materials, cost, and otherfactors may affect the choice of material used to fabricate a cuvette600. As discussed above, the presence of structures 604, theavailability of compression (e.g., as may be applied via a structure610, or directly to at least a portion of a support structure 606 andcover portion 612), and other factors, may allow the use of materialsthat may be less rigid than quartz, for example, yet may still providethe requisite optical and mechanical properties for use in the systemsand methods disclosed herein. In addition, the presence of structures604, the availability of compression, and other factors, may allow theuse of manufacturing techniques and tolerances that might otherwise notbe possible (e.g., due to the possibility of deformation or otherfactors) in the absence of such structure, compression, and otherfactors. In addition, the presence of structures 604, the availabilityof compression, and other factors, may allow the use of materials,including less costly materials, than might otherwise be used in theabsence of such structure, compression, and other factors.

Thus, proper design, construction, and materials for support structures604 and base portions 606 are important for cuvettes 600 and their use.

In some embodiments, these controlled thickness areas 613 (see, e.g.,FIGS. 8A, 8B, and 8D) are selected to be positioned over the analysisareas 608. In some embodiments, these controlled thickness areas 613 canimpart certain optical properties over or near the analysis areas. Someembodiments may configure the structures 604 to also impart opticalproperties on light passing through the cuvette 600. Optionally, in someembodiments, the structures 604 may be configured to not have an impacton the optical qualities of the cuvette 600. In such an embodiment, thestructures 604 may be configured to have one or more optically absorbentsurfaces. For example and without limitation, certain surfaces may beblack. Optionally, some embodiments may have the structures 604 formedfrom a material to absorb light. Optionally, the structures 604 can bepositioned to provide mechanical support but do not interact with theoptical properties of cuvette 600 near the analysis areas.

For example, certain surfaces, including a surface 614 of a controlledthickness area 613, and a surface 618 of a structure 604, may be coatedwith a black, or other color, coating. Such a coating may include onelayer, and may include multiple, layers. For example, suitable coatingsof a surface 614 or 618 may include 2, 3, 4, 5, 6, 7, or more layers. Inembodiments, e.g., a surface of a structure 604 (e.g., a surface 618) ora surface 614 may be covered by 3 or 5 layers of coating. Such a coatingmay include a dye, an ink, a paint, a surface treatment, a colored tape,or other coating or surface treatment. In embodiments, a black or othercolor marker (e.g., a Paper Mate®, or Sharpie®, or Magic Marker®, orother marker) may be used to coat a surface 614 of a controlledthickness area 613 or a surface 618 of a structure 604. For example, anextra-large black marker may be used to apply multiple coats of blackink to a surface 614 or to the outer surface 618 of a structure 604 toprovide an optically absorbent surface and so to improve the opticalqualities of a cuvette 600. In embodiments, a surface 614 or 618 may becoated or treated so as to affect or reduce reflectance (whether PIR orTIR) at the surface. A reduction in reflectance at a surface may affect(e.g., reduce) background illumination from a surface.

In embodiments, certain surfaces, including a surface 614 of acontrolled thickness area 613, and a surface 618 of a structure 604, maybe coated or covered with a material which enhances reflectance at thesurface. Reflectance at a surface may be increased, for example, bycoating a surface, or attaching a material to a surface; suitablematerials for increasing reflectance include aluminum, silver, gold, anddielectric materials (e.g., magnesium fluoride, calcium fluoride, orother salt or metal oxide; or other reflective or dielectric material).Such a coating or covering may include one layer, and may includemultiple, layers. For example, suitable coatings and coverings of asurface 614 or 618 may include 2, 3, 4, 5, 6, 7, or more layers. Anincrease in reflectance at a surface may affect (e.g., increase)trans-illumination from a surface. An increase in reflectance at asurface may aid or enhance imaging of a sample within an analysis area608, or may aid or enhance optical analysis of a sample within ananalysis area 608.

It should be understood that the cuvette 600 is typically formed from anoptically transparent or optically transmissive material. Optionally,only select portions of the cuvette 600 (such as, e.g., the analysisareas or areas associated with the analysis areas) are opticallytransparent or optically transmissive. Optionally, select layers orareas in the cuvette 600 can also be configured to be non-lighttransmissive. A portion or area of a cuvette may be covered or coated soas to be light absorbing; for example, a surface (or portion thereof)may be coated with a dark, or a light-absorbing, dye or ink. In afurther example, a surface (or portion thereof) may be covered with adark, or a light-absorbing, coating, such as a dark or light-absorbingmaterial, e.g., tape, or cloth, or paper, or rubber, or plastic.

FIGS. 6A, 6B, and 8A-8D illustrate embodiments in which the cuvette 600rests on a base support 620 wherein some or all of the base support 620is formed from an optically transparent or transmissive material. Insome embodiments, the optically transparent or transmissive portions areconfigured to be aligned with the analysis areas of the cuvette 600 toallow for optical interrogation of the sample in the analysis area. Inone non-limiting example, the base support 620 can be movable in the X,Y, or Z axis to move the cuvette 600 to a desired position for imaging.In some embodiments, the base support 620 comprises a platform or stagethat moves only in two of the axes. Optionally, some support structuresmay move only in a single axis. The cuvette 600 can be configured to beoperably coupled to the support structure 600 through friction,mechanical coupling, or by retaining members mounted to one or both ofthe components. In embodiments, compression, or other force may beapplied to a cuvette 600 or a base support 620, or both, in order toensure adequate contact and proper fit between a cuvette 600 and a basesupport 620. In embodiments, such compression may aid in ensuring thatan optically transmissive surface of a cuvette 600, or of a base support620, or such surfaces of both, is optically flat and substantially freeof distortion. For example in embodiments, a cuvette 600 may be pressedagainst a base support 620 in order to reduce or obviate any possibleoptical distortion which might be caused by imperfections orabnormalities in an optical surface of a cuvette 600. In embodiments,such force (e.g., compression) may aid in providing desired opticalproperties, effective to allow passage of light with distortion at theinterface than might otherwise be produced. In embodiments, such force(e.g., compression) may be applied, at least in part, via a structure610 or via multiple structures 610.

FIGS. 6A, 6B, 8A, 8B, 8C, and 8D further show embodiments in whichillumination for dark field or brightfield observation may be providedby an illumination source 650 (such as but not limited to a ringlight asshown) placed below the base support 620 to locate illuminationequipment below the level of the cuvette 600. This configuration leavesthe upper areas of the cuvette 600 available for pipettes, samplehandling equipment, or other equipment to have un-hindered access toopenings or other features on a top surface of the cuvette 600.Optionally, some embodiments may locate an illumination source 660(shown in phantom) above the cuvette 600 to be used in place of, insingle, or in multiple combination with underside illumination (e.g., anunderside illumination source 650 as shown). An objective 670 can bepositioned as shown, or in other configurations, to observe the samplebeing illuminated. It should be understood that relative motion betweenthe cuvette 600 and the optical portions 650 and 670 can be used toallow the system to visualize different analysis areas in the cuvette600. Optionally, only one of such components is placed in motion inorder to interrogate different areas of the cuvette 600.

Referring now to FIG. 7A, one embodiment of a suitable imaging systemwill now be described in more detail. FIG. 7A shows a schematiccross-sectional view of various components positioned below the basesupport 620. The cross-section is along the area indicated by bentarrows 7 in FIG. 6A.

FIG. 7A shows an embodiment in which the cuvette 600 comprises a baseportion 606 and analysis areas 608 defined by a cover portion 612.Optionally, the analysis areas 608 may be defined within a single piece.Optionally, the analysis areas 608 may be defined by using more than twopieces, such as but not limited a discrete cover piece for each of theanalysis areas 608. In one embodiment, the layer 606 comprises opticallyclear plastic such as but not limited to cyclo-olefin polymerthermoplastic which deliver superior optical components andapplications. In some embodiments, one or more layers or components maybe formed from glass, acrylic, clear polymer, or other transparentmaterial. The cuvette 600 illustrated in FIG. 7A includes five separateanalysis areas 608; these areas are shown in cross-section in thefigure; analysis areas 608 having such a cross-section may berectangular, or square, or other shape. For example, analysis areas 608may comprise elongated channels providing shallow chambers withrelatively large amounts of surface area though which samples may beobserved. In embodiments, analysis areas 608 may have curved, orpolygonal, or irregular shapes, and may be separate, or may be connectedby connecting channels. It will be understood that a cuvette 600 mayinclude a single analysis area 608; or may include two analysis areas608; or may include three analysis areas 608; or may include fouranalysis areas 608; or may include five (as shown in FIG. 7A) or moreanalysis areas 608.

In embodiments, a channel in a cuvette 600, such as an analysis area608, may have an irregular shape so that a cross-sectional dimensiondiffers along the length of the channel; for example, a channel in acuvette 600 may have a narrow end portion and a wider middle portion. Inembodiments, a channel in a cuvette 600, such as an analysis area 608,may have U-shape or other shape in which a first elongated portion of asingle analysis area is disposed near to, or alongside, a secondelongated portion of the same analysis area 608. For example, in such anembodiment, the rectangle indicated by the lead line labeled “608” inFIG. 7A may be a portion of same analysis area illustrated by therectangle immediately to the left of the rectangle indicated by the leadline labeled “608”.

In embodiments, a sample to be interrogated can be held in whole or inpart in an analysis area 608. In embodiments, more than one portion of asample, or more than one sample, or portions of more than one sample,may be held in an analysis area 608. In embodiments, portions of asample, or portions of different samples, within a channel of a cuvettetoo, e.g., within an analysis area 608, may be separated by an airbubble, or by an oil droplet, or by another material or materials.

In embodiments, analysis of a sample held in an analysis area 608 maycomprise optical observation, measurement, or imaging of at least aportion of an analysis area 608. In embodiments, optical observation,measurement, or imaging of at least a portion of an analysis area 608may comprise optical observation, measurement, or imaging of an entireanalysis area 608. In embodiments, analysis of a sample held in ananalysis area 608 may comprise optical observation, measurement, orimaging of only a portion of an analysis area 608. In embodiments,analysis of a sample held in an analysis area 608 may comprise opticalobservation, measurement, or imaging of a region of interest (ROI)within at least a portion of an analysis area 608. In embodiments,analysis of a sample held in an analysis area 608 may comprise opticalobservation, measurement, or imaging of multiple ROIs within an analysisarea 608. For example, where a channel in a cuvette 600 has a narrow endportion and a wider middle portion, multiple ROIs may be observed,measured, or imaged in the wider middle portion, while, for example,only a single ROI (or no ROI) may be observed, measured, or imaged inthe narrower end portion.

By way of non-limiting example, the optics below the base support 620may include a ringlight 650 that comprises a toroidal reflector 652 anda light source 654. Other illumination components suitable for darkfield illumination may be used; thus the optics may include othersources of illumination, alone or in combination with such a ringlight.Some embodiments may use a mirror. Some embodiments may use a coatedreflective surface. Some embodiments may use a different reflector thanthe ones shown in the figure (e.g., may not use toroidal reflection inilluminating a sample). Some embodiments may use a parabolic reflector.Some embodiments may use a parabolic reflector in the shape of anelliptic paraboloid. Some embodiments may use a plurality of individualreflector pieces. Some embodiments may not use any reflector. Someembodiments obtain oblique illumination through the use of angled lightsources positioned to direct light with or without further assistancefrom one or more external reflectors.

Multiple wavelengths of light may be emitted by a light source or lightsources, either simultaneously or sequentially. The embodimentillustrated in FIG. 7A shows excitation energy sources 680, 682, and 684such as but not limited laser diodes at specific wavelengths that aremounted to direct light into the sample in analysis area 608. In onenon-limiting example to facilitate compact packaging, the energy sources680, 682, and 684 may direct light to a dichroic element 690 (e.g., adichroic mirror or beamsplitter) that then directs the excitationwavelengths into the analysis area 608. The excitation wavelength(s)cause fluorescence wavelengths to be emitted by fluorophores inmarker(s), dye(s), or other materials in the sample. The emittedfluorescence wavelengths are funneled through the objective 670, throughthe dichroic element 690, through an optional filter wheel 692, and intoa detector 700 such as but not limited to a camera system. By way ofnon-limiting example, the dichroic element 690 is configured to reflectexcitation wavelengths but pass fluorescence wavelengths and anywavelengths desired for optical observation.

Multiple wavelengths of light may be acquired either simultaneously orsequentially. In one embodiment, all fluorescence excitation wavelengthsilluminate the sample in analysis area 608 simultaneously. For example,a detector 700 may be coupled to a programmable processor 710 that cantake the captured signal or image and deconstruct which wavelengths areassociated with which fluorophores that are fluorescing. In someembodiments, excitation sources may illuminate the sample sequentiallyor in subsets of the entire number of excitation sources. Of course, itshould be understood that the system is not limited tofluorescence-based excitation of fluorophores in a sample, and thatother detection techniques and excitation techniques may be used inplace of, or in single or multiple combination with fluorescence. Forexample, some embodiments may also collect dark field illuminationscatter information simultaneously or sequentially in combination withfluorescence detection.

In a further embodiment, illumination of a sample is accomplished over aperiod of time by scanning a spot, or spots, of light, over the sample(e.g., within an analysis area 608 or within an ROI within, orcomprising, an analysis area 608). Such a spot, or spots, may comprisepoints of light, or may comprise lines of light, or may comprise othershapes, or may comprise combinations thereof. Such a scan may be, e.g.,a raster scan (e.g., where illuminated regions form a series of adjacent(dotted or dashed) lines), a rectangular scan (e.g., where illuminatedregions form nested square or rectangular shapes delimited by (dotted ordashed) lines), a spiral scan (e.g., where illuminated regions form a(dotted or dashed) spiral line pattern), or other shape or pattern scan.

Similarly, examination of a sample may be accomplished at one time, ormay be accomplished over a period of time by measuring light from aspot, or spots, of light, over the sample (e.g., within an analysis area608 or within an ROI within, or comprising, an analysis area 608). Suchmeasurements may be recorded. Such a spot, or spots, may comprise pointsof light, or may comprise lines of light, or may comprise other shapes,or may comprise combinations thereof. Such a scan may be, e.g., a rasterscan (e.g., where illuminated regions form a series of adjacent (dottedor dashed) lines), a rectangular scan (e.g., where illuminated regionsform nested square or rectangular shapes delimited by (dotted or dashed)lines), a spiral scan (e.g., where illuminated regions form a (dotted ordashed) spiral line pattern), or other shape or pattern scan.

Such scanning (whether for illumination, measurement, or both) may beaccomplished, for example, by use of piezoelectric, electromechanical,hydraulic, or other elements operably connected to, e.g., opticalelement 690, a mirror or mirrors (e.g., a mirror associated withexcitation energy sources 680, 682, or 684), or to other reflectors,gratings, prisms, or other optical elements.

Light scattered by an object in a sample within a sample holder (e.g., acell, or a bead, or a crystal) will be scattered at a plurality ofscatter angles, where a scatter angle may be measured with respect to aray of incident light passing from a light source to the object. Such aplurality of scatter angles comprises a range of scatter angles. Such asample holder may have features as disclosed herein, and may beconfigured to provide pathways for internal light reflections. Anobjective lens configured to image the object will gather and focus thescattered light, where the light may be passed to a detector. Such lightfocused by an objective lens and focused on a detector may form a spotof light on the detector. In embodiments, the light passing from theobjective lens to the detector may be focused by a further lens; suchfocusing may reduce the size of the spot of light formed on thedetector. The light focused on a detector, whether or not it passesthrough a further lens, will comprise light scattered at a plurality ofscatter angles from the object within the sample holder.

Applicants disclose herein methods, systems, and devices (e.g., sampleholders) which allow detection of a smaller range of scatter angles thanotherwise possible, thereby providing greater resolution and betterimaging of samples and of objects within a sample. Applicants discloseherein design features for cuvettes which may be used to control theangles and intensities of light rays incident on the sample, e.g., viaPIR and TIR, effective to control the angles at which scattered light ismeasured.

Due to constraints imposed by non-imaging optics of many systems (e.g.etendue, or the extent of the spread of light passing through thesystem) the scatter angles of light arriving at a detector can be widerthan desired. For example, in some ringlight-cuvette combinations usingLEDs as light sources, light rays striking the sample may be spread outto at least 20 degrees around the principal angle. In other words, ifthe principal ray strikes the sample at 60 degrees, the other rays ofthe bundle of light rays may strike the sample at scatter angles ofabout 50 degrees to about 70 degrees. It will be understood that thespread of the cone of scatter angles of light collected by an objectivedepends upon the numerical aperture of the lens. In such a case, thelight collected by the objective lens (e.g., having a numerical apertureof 40 degrees) would be in a cone of about 30-70 degrees. Consequently,light scattered over a wide range of scatter angles will arrive at thedetector; for example, such a system will measure all the lightscattered by the sample in a large cone centered around 60 degrees+/−40degrees. However, as disclosed herein, some applications requiredetection of light within a narrower range of scatter angles, e.g.,within a very narrow range of angles (say 60+/−5 degrees). Applicantsdisclose herein that, in order to provide light measurements from withinthis narrower range, an aperture can be placed in the Fourier (or backfocal plane) of the objective lens (or any plane conjugate with thisplane). In the Fourier plane, the angle information is spatially coded.Therefore, depending upon the shape and size of this aperture, lightcoming from the sample at specific angles can be prevented from reachingthe detector (e.g., blocked or filtered out). An annular aperture willblock or filter out the inner angles (say 60+/−30 degrees). Theresultant measurement can therefore be tailored to the desired angles.

In embodiments, an aperture may be provided through which light from anobjective lens passes prior to contacting a detector. In embodiments, anaperture may be provided through which light from a further lens (afterpassing through an objective lens) passes prior to contacting adetector. Where such an aperture is configured to limit the light whichpasses through to the detector, the light which passes through will bewill be reduced to light from fewer scatter angles, and to light from asmaller range of scatter angles, than the light which passes through inthe absence of such an aperture. In embodiments, such an aperture maycomprise a single hole, such as a circular hole. In embodiments, such anaperture may comprise a single annulus, such as a circular ring throughwhich light may pass, and having a central area (e.g., a circular area)through which light does not pass. In embodiments, such an aperture maycomprise two, or three, or more, concentric annuli through which lightmay pass, and may include a central area (e.g., a circular area) throughwhich light does not pass. In embodiments, such an aperture may comprisea shape other than a circular or annular shape.

Such an aperture disposed between an objective and a detector, e.g.disposed between a further lens and a detector (where light passesthrough an objective lens prior to passing through the further lens),provides the advantage of sharper discrimination of the light scatteredfrom the sample, improving the resolution of light-scatter images (e.g.,dark field images) obtained from the sample. In embodiments where lightintensity may be a factor, the intensity of light applied (e.g., from alight source, or from multiple light sources) may be increased inconfigurations having an aperture as disclosed herein, as compared toconfigurations lacking an aperture as disclosed herein.

A system may include a sample holder having features as discussed anddescribed herein, and light sources, dichroic mirrors, and otherelements as shown in FIG. 7A. As illustrated in FIG. 7B, systems havingsimilar features (e.g., similar to those shown in FIG. 7A and otherfigures herein) may include a sample holder 600, a light source 650(e.g., light sources 654, or an excitation source 680, or both), anobjective lens 670, an aperture 694, a further lens 696, and a Fourierlens 698. An aperture 694 may have a single passage for allowing lightto pass thorough to a detector 700. A detector 700 may be operablylinked to a processor (e.g., a programmable processor) 710. Inembodiments, an aperture 694 may comprise two passages for allowinglight to pass thorough to a detector 700. In embodiments, an aperture694 may comprise three passages for allowing light to pass thorough to adetector 700. In embodiments, an aperture 694 may comprise four, ormore, passages for allowing light to pass thorough to a detector 700. Inembodiments, a passage in an aperture 694 may comprise a circular holeallowing light to pass thorough to a detector 700. In embodiments, apassage in an aperture 694 may comprise two, or three, or four or morecircular holes allowing light to pass thorough to a detector 700. Inembodiments, a passage in an aperture 694 may comprise an annulusconfigured to allow light to pass thorough to a detector 700, and mayinclude a central portion which does not allow light to pass through toa detector 700. In embodiments, a passage in an aperture 694 maycomprise two or more annuli (e.g., in embodiments, concentric annuli)each of which is configured to allow light to pass thorough to adetector 700; and such an aperture 694 may include a central portionwhich does not allow light to pass through to a detector 700. Such anannulus, and such annuli, may have a circular, or elliptical, or otherannular shape.

Accordingly, Applicants disclose systems for imaging a sample,comprising: a sample holder, a light source for illuminating an objectheld within said sample holder, an objective lens configured to collectand focus light scattered from an object held within said sample holder,wherein said scattered light comprises light scattered at a plurality ofscatter angles, an optical aperture for passing light from saidobjective lens, and a further lens configured to focus light from saidobjective lens onto said optical aperture, wherein said optical apertureis configured to allow only a portion of light focused by said objectivelens to pass through the aperture, whereby said portion of light allowedto pass through said aperture consists of light scattered at only aportion of said plurality of scatter angles.

As used herein, the terms “epi” and “epi-illumination” refer toillumination of a sample by light traveling in a direction that isgenerally away from an objective or other optical element used toobserve or image the sample. Thus, in the absence of fluorescence, animage of a sample illuminated by epi-illumination is formed with lightreflected or scattered from the sample (light travels from the lightsource to the sample, and is reflected or scattered by the sample backto the optical elements for observation, imaging, or measurement). Asused herein, the terms “trans” and “trans-illumination” refer toillumination of a sample by light traveling in a direction that isgenerally towards an objective or other optical element used to observeor image the object (light travels from the light source to and throughthe sample, and continues on to the optical elements for observation,imaging, or measurement). Thus, in the absence of fluorescence, an imageof a sample illuminated by trans-illumination is formed with lightpassing through, or scattered by, the sample.

Where a light source is disposed on the same side of a sample as theobjective or other optical elements used to observe or image a sample,light from the light source travels directly to the sample, and thesample is thus typically observed or imaged by epi-illumination.However, even where a sole light source is placed on the same side of asample as the objective or optical elements, a sample holder asdisclosed herein is able to provide trans-illumination of a sample inaddition to epi-illumination. Thus, both directions of illumination areenabled without requiring placement of light sources on both sides of asample. Such a configuration is compact, sparing of resources, and,since the light source and other optical elements are disposed on onlyone side of the sample holder, the configuration allows unimpeded accessto the side of the sample holder without interference by the opticalelements. Thus, such a configuration provides the advantage of enablingloading, mixing, and removal of a sample and reagents in the sampleholder without interference with optical imaging or measurements, or theapparatus and elements used for optical imaging or measurements.

As illustrated by the images shown in FIGS. 4A and 4B, addingtrans-illumination to dark field images greatly enhances the image andgreatly enhances the information available from the image. The methodsand systems disclosed herein provide such greatly enhanced images bycombining both epi-illumination and trans-illumination, usingillumination from a single direction, and, in embodiments, from only asingle light source.

As disclosed herein, a sample holder such as a cuvette 600 (e.g., asillustrated in FIGS. 8A-8D) is configured to allow internal reflectionof light from a light source (whether PIR or TIR), so that a sample heldin an analysis area 608 of a cuvette 600 is illuminated by direct light(epi-illumination; e.g., light travelling along path 830) and is alsoilluminated by indirect, reflected light (trans-illumination; e.g.,light travelling along a path 820 or 825). As disclosed herein, lightfrom a light source disposed on the same side of a cuvette 600 asoptical elements 670, 690, 700, etc., may provide both epi- andtrans-illumination of a sample.

Referring now to FIGS. 8A-8D, a still further embodiment will now bedescribed. FIGS. 8A-8D show a schematic of a cross-section of a portionof a cuvette 600 and the dark field scatter illumination source such asbut not limited to the ringlight 650 shown in FIGS. 6A and 6B. Basesupport 620 is also shown in FIGS. 8A-8D. FIGS. 8A-8D include bracketsand arrows to indicate structures or portions of structures; forexample, the bracket labeled 600 indicates the entire cuvette 600 shownin the figure; the bracket labeled 612 indicates the cover portion 612of the cuvette 600. The arrows 621 to 626 in FIG. 8A indicate dimensionsfor the indicated portions of the cover portion 612. It will beunderstood that these dimensions may vary in different embodiments of acuvette 600, and that such variations may depend upon the size,application, materials, optical wavelengths, samples, and other elementsand factors related to the construction and use of a cuvette 600. Forexample, in embodiments, the distance 621 between support structures 604may be between about 0.1 millimeter (mm) to about 1 centimeter (cm), andin embodiments may be between about 1 mm to about 100 mm, or betweenabout 1.5 mm to about 50 mm, or between about 2 mm to about 20 mm. Infurther embodiments, the distance 621 between support structures 604 maybe between about 0.5 mm to about 10 mm, or between about 1 mm to about 5mm. In embodiments, the height 622 of a support structure 604 may bebetween about 0.1 mm to about 100 mm, or between about 0.5 mm to about50 mm, or between about 1 mm to about 25 mm. In further embodiments, theheight 622 of a support structure 604 may be between about 0.1 mm toabout 10 mm, or between about 1 mm to about 5 mm. Similarly, inembodiments, the height 623 of a controlled thickness area 613 may bebetween about 0.1 mm to about 100 mm, or between about 0.5 mm to about50 mm, or between about 1 mm to about 25 mm. In further embodiments, theheight 623 of a controlled thickness area 613 may be between about 0.1mm to about 10 mm, or between about 1 mm to about 5 mm. In embodiments,the thickness 624 of a layer 800 may be between about 0.01 mm to about10 mm, or between about 0.05 mm to about 1 mm, or between about 0.1 mmto about 0.5 mm. In embodiments, the width 625 of an analysis area 608may be between about 0.05 mm to about 100 mm, or between about 0.5 mm toabout 50 mm, or between about 1 mm to about 25 mm. In furtherembodiments, the width 625 of an analysis area 608 may be between about0.1 mm to about 10 mm, or between about 1 mm to about 5 mm. Inembodiments, the width 626 of a support structure 604 may be betweenabout 0.1 mm to about 100 mm, or between about 0.5 mm to about 50 mm, orbetween about 1 mm to about 25 mm. In further embodiments, the width 626of a support structure 604 may be between about 0.05 mm to about 10 mm,or between about 0.5 mm to about 5 mm.

It will be understood that optical components and arrangements forillumination, for excitation, for observation of emission, and the like,as illustrated in any one figure herein, may suggest components andarrangements that may be applied in embodiments of other figures, evenif such particular components or arrangements are not explicitly shownin each figure. For example, although a ringlight 650 or other source ofillumination 650 is not included in FIG. 8D, in any of the embodimentsshown, and in other embodiments, a ringlight 650 or other source ofillumination 650 (see, e.g., FIGS. 8A, 8B, and 8C) may be used toilluminate the analysis area 608 (analysis area 608 is shown in FIGS. 8Aand 8B). As examples of optical components which are suitable for usewith a cuvette 600, ringlight components 652 and 654 are shown in FIGS.8A, 8B, and 8D; in embodiments, other, or other numbers of, illuminationcomponents may be used. For example, light source 654 may be white lightor light sources such as but not limited to light emitting diodes (LEDs)or laser diodes with specific wavelength output or output ranges.Optionally, the ring of light source 654 could be fiber optic cableconfigured to provide a ring of light (e.g., with many splices).Optionally, the light source 654 may be an LED which has a specificnarrow divergence angle controlled by the reflector. It may be desirableto control the divergence angle from a ringlight through the selectionof the light source or through the design of the reflector.

By way of non-limiting example, a light source 654 may use laserillumination to provide a narrow light pattern, resulting in lowertrans-illumination background in the present epi-style lightingconfiguration (where illumination components are all on one side of thesample) because the light source: provides a narrow spot of light(directed within the sample analysis area 608); provides light of narrowspectral width (e.g., light of wavelengths within a narrow rangecentered around a particular main wavelength); and is a coherent source.Optionally, use of a LED as the illumination source 654 may also providea small spot size (e.g., a small spot size within an analysis area 608)and so provide some of the beneficial properties achieved by a laserlight source. For these, and other reasons, a laser light source (or anLED providing a small spot size) is effective to lower background signallevels as compared with other illumination configurations. Laserillumination may reduce scattered light as compared to that whichtypically occurs with more diffuse light sources, and so may reduce thebackground in one channel (e.g., within a first analysis area 608) byreducing light scattered into that channel from an adjacent channel(e.g., from an adjacent, second analysis area 608). Thus, laserillumination can result in less trans-illumination background than wouldbe expected from illumination by more diffuse light sources. Of course,it is desirable that the decrease in trans-illumination is less than thedecrease in background, where the more significant drop in backgroundresults in a more distinguishable signal. Optionally, use of a LED asthe illumination source 654 provides a diffuse light pattern, withincreased background and increased trans-illumination. Of course, it isdesirable that the increase in trans-illumination is greater than theincrease in background.

Some cuvette embodiments may include cuvettes formed from a plurality ofindividual layers adhered together, having the cuvette molded from oneor more materials, or having reflective layers added to the cuvette atdifferent surfaces to enhance single or multiple internal reflections(e.g., to enhance TIR or PIR).

In embodiments, systems, cuvettes, and optical elements disclosed hereinmay be operating in combination with fluorescence, it may be desirablethat dark field illumination used with such systems and cuvettes not bewhite light illumination. However, some embodiments may use just whitelight, e.g., if fluorescence detection is not used in combination withdark field or brightfield microscopy.

FIGS. 8A and 8B shows that in some embodiments, the device may havelayers in the cuvette 600 that are optically non-transmissive such aslayer 800. This may be useful in embodiments where the light source 654is diffuse and light is not directed to specific locations. The layer800 can block light that is not entering the cuvette 600 at desiredangles or locations. The layer 800 can be configured to be positioned toprevent illumination except through the area below the analysis areas608. Some may only have specific areas that are blacked out nearest theanalysis areas 608. Some embodiments may have blacked out ornon-tranmissive material in more than one layer. Some may have blackedout or non-transmissive material in different orientations, such as butnot limited to one being horizontal and one being vertical ornon-horizontal.

It will be understood that, in embodiments, a layer 800 may be opticallytransmissive. For example, FIG. 8D presents an embodiment in which alayer 800 is optically transmissive. In some embodiments, a layer 800may comprise an optically transmissive material having an index ofrefraction that is different than the index of refraction of acontrolled thickness area 613, or of a base support 620, or of both. Insome embodiments, a layer 800 may comprise an optically transmissivematerial having an index of refraction that is the same as the index ofrefraction of a controlled thickness area 613, or of a base support 620,or of both.

In FIGS. 8A, 8B, and 8C, a light source is shown located below a cuvette600 (near to optics 652 and 654) and provides light directed from belowbase portion 606. Such a light source may be understood to be in placein the example illustrated in FIG. 8D as well. As shown in thesefigures, a light source 650 may include a ringlight 654 and a toroidalreflector 652. Other elements, including without limitation lenses,filters, gratings, mirrors and other reflective surfaces, opticalfibers, prisms, and other elements may be included. In embodiments, alight source may comprise a laser, or a LED, or other light source; andmay comprise a fiber optic which carries light from such a source toanother location, or which directs light towards an optical element. Onedesign criterion for optical systems is the divergence, or divergenceangle, of light from the light source; a light beam of width D with lowdivergence provides a smaller spot at a given distance from the sourcethan does a light beam of width D with high divergence. In general, alight source 650 which provides light with low divergence is preferred.Such optical elements and configurations may be designed so as toprovide light which is substantially collimated, e.g., most or all lightis directed along substantially parallel paths towards the sample (e.g.,towards an analysis area 608). However, in embodiments where diffuse orscattered light is preferred, a light source 650 with high divergencemay be used.

As shown in FIG. 8C, an embodiment of an optical system suitable as partof device or system as disclosed herein may include optics (e.g., alight-source 650, e.g., as shown in FIG. 8C as a ringlight 654, and anobjective 670), a cuvette 600, and a base support 620 configured to holdand position a cuvette for imaging. In embodiments as shown in FIG. 8C,a base support 620 may include optical features 802 configured torefract (or diffract, or otherwise alter the path of) light from alight-source 650. As illustrated in FIG. 8C, optical features 802 maycomprise an array of lenslets. It will be understood that opticalfeatures 802 may comprise any suitable optical feature. In embodiments,optical features 802 may comprise lenslets, or diffraction gratings, orFresnel lenses, or convexities, or concavities, or other shapes andfeatures which may refract, diffract, or otherwise alter light, orcombinations thereof. In embodiments, such optical features 802 maycomprise different material than base support 620, and may have adifferent index of refraction than base support 620. For example, lightaffected by optical features 802 may be directed towards an analysisarea 608, either directly, or indirectly via reflection (e.g., internalreflection) suitable for use in methods disclosed herein, e.g., so as toprovide both epi-illumination and trans-illumination of a sample in ananalysis area 608. As illustrated in the embodiment shown in FIG. 8C,such embodiments may also include a light path which bypasses opticalfeatures 802. Such a light path may be better suited for imaging of asample within an analysis area 608 than paths which would requireimaging through an optical feature 802. In embodiments, both types oflight paths (i.e., bypassing optical features 802 and passing throughoptical features 802) may be provided at the same time, thus providingsuitable optics for image analysis of a sample illuminated by bothepi-illumination and trans-illumination from a light source situated onthe same side of a cuvette 600 as a light source 650.

The cuvette 600 includes features which affect the path of lightilluminating the cuvette and the sample within the cuvette. Suchtrans-illumination may be effected by light reflected within a cuvette600 (e.g., by internal reflection, including or primarily by partialinternal reflection (PIR) or total internal reflection (TIR) from, forexample, a surface 612, a surface 604, or other surfaces or combinationsof surfaces. Other examples of pathways of light undergoing TIR areshown, for example, in FIGS. 8A, 8B, and 8D.

As illustrated in FIG. 8D, in embodiments, a cuvette 600 of an opticalsystem of a device or system as disclosed herein, and suitable for usein methods disclosed herein, may include features which affect the pathof light illuminating internal portions of the cuvette 600, such aslight illuminating an analysis area 608, and the sample within ananalysis area 608 of a cuvette 600. As shown in FIG. 8D, a layer 800 mayinclude features which refract, diffract, or otherwise affect or alterthe path of light entering an analysis area 608. Such alteration oflight paths may affect, and may improve, the illumination of samplewithin an analysis area 608. In the example shown in FIG. 8D, lightenters layer 800 from a transverse direction; the light paths arealtered by the shape (and material properties) of the layer 800, and aredirected as desired into analysis area 608. For example, an externalsurface of a layer 800 may be flat (e.g., external surface 674) or maybe curved (e.g., external surface 676). For example, an internal surfaceof a layer 800 may be flat (not shown in FIG. 8D; see, however, suchsurfaces in FIGS. 8A and 8B (although layers 800 in FIGS. 8A and 8B arenot optically transmissive, these surfaces are shown as being flat) ormay be curved (e.g., internal surface 678 shown in FIG. 8D). Inembodiments, such alteration of light paths is effective to provide bothepi-illumination and trans-illumination of samples in an analysis area608.

FIGS. 8A, 8B, 8C, and 8D illustrate light paths within a sample holderproviding examples of TIR and PIR within a cover portion 612 at an uppersurface 614 or at surface 618 in a support structure 604. A sampleholder, such as a cuvette 600, may have an optically transmissivesurface through which light may pass; in embodiments, such an opticallytransmissive surface may allow light to pass without significantdistortion or diminution in light intensity. A sample holder, such as acuvette 600, may be made of optically transmissive material, effectivethat light may pass within the sample holder. In embodiments where asample holder is at least partially made of optically transmissivematerial, light may pass through an optically transmissive surface of asample holder, and may travel within the sample holder. In embodiments,light traveling within a sample holder may be reflected at one or moresurfaces, and travel along a reflection path within a sample holder.Where light from a light source disposed outside a sample holder entersa sample holder through an optically transmissive surface of a sampleholder, such light may travel within the sample holder away from thelight source, and may be reflected at a surface of the sample holder, sothat the reflected light may travel in a direction towards the lightsource after being reflected. Such reflections may be by PIR or TIR.

That is, light passing within a cuvette 600 may reflect off a surface(e.g., a surface 614 or surface 618 as shown in FIGS. 8A and 8B). Suchinternal reflections may be effective to illuminate a sample within ananalysis area 608 with indirect light; in combination with directillumination (where the light is not reflected prior to impinging on asample), the sample may in this way receive epi-illumination(illumination from the same side as the optical detection elements) andtrans-illumination (illumination from the side opposite the opticaldetection elements). Where a surface 614, or a surface 618, or both, areconfigured to absorb light (e.g., are painted or coated black), anepi-illumination source alone may be used to provide dark field images.Where a surface 614, or a surface 618, or both, are configured toscatter light (e.g., are not polished or have rough surfaces), anepi-illumination source alone may be used to provide such scatteredlight suitable for obtaining bright-field images.

It will be understood that light wavelengths, material, surfaces, andconfigurations that promote or enhance PIR may not be suitable oreffective to promote or enhance TIR. It will be understood that lightwavelengths, material, surfaces, and configurations that promote orenhance TIR may not be suitable or effective to promote or enhance PIR.Thus, there are designs and constructions where one or the other of PIRand TIR may be promoted, in the absence of the other. In embodiments,there are designs and constructions where both of PIR and TIR may bepromoted. In embodiments, there are designs and constructions in whichneither PIR nor TIR are promoted.

As illustrated in FIG. 8A, support structures 604 may have rectangularor square cross-sections. It will be understood that a support structure604 may have a cross-sectional shape other than square or rectangular;for example, as shown in FIG. 8B, a support structure 604 may have atriangular cross-sectional shape; other cross-sectional shapes (e.g.,rounded or semi-circular, or jagged, or irregular) may also be suitablefor use with systems and cuvettes disclosed herein. PIR and TIR aretunable features that can selected based on the material used for thecuvette 600, any coatings, cladding, or coverings applied, and thegeometry or thickness of the controlled thickness area 613 of thecuvette 600. In embodiments, PIR may be preferred, and light, materials,and configurations may be selected to enhance PIR.

In embodiments, TIR may be preferred. In embodiments, the wavelength orwavelengths of light from a light source 650 may be selected to enhanceTIR. In embodiments, the material, thickness, surface configuration, andother features of a cuvette 600 may be selected to enhance TIR. Forexample, the height (as measured from the base of cover portion 612 incontact with layer 800) of the controlled thickness area 613 will affectthe angle and intensity of light reflected by TIR that arrives atanalysis area 608. Configuration of a cuvette 600 so as to enable TIR oflight within the cuvette which allows for oblique angle illumination ofa sample (illumination coming from above the sample) is desirable,particularly for dark field microscopy. In some embodiments, it isdesirable to maximize TIR from above the sample. Optionally, in someembodiments a cuvette 600 may be configured to provide TIR only fromsurfaces over the analysis areas 608. Optionally, some embodiments maybe configured to provide TIR only from surfaces over the controlledthickness area 613 (e.g., in the embodiments shown in FIGS. 8A and 8B,generally above analysis area 608). Optionally, in some embodiments, acuvette 600 may be configured so as to provide TIR of light from othersurfaces in the cuvette 600; for example, TIR of light from othersurfaces in the cuvette 600 may be provided so as to scatter light atoblique angles, effective that the light is directed back to theanalysis area 608.

The design and materials used to construct a cuvette 600 may be selectedand configured so as to provide TIR of light. For example, in someembodiments, configurations which provide TIR, or which provideincreased or enhanced amounts of TIR, include, without limitation:configurations in which the dimensions of controlled thickness area 613are compatible with, or which promote, TIR; configurations in which theangle or angles of a surface 614 or a surface 618 (e.g., with respect toincident light) are compatible with, or which promote, TIR;configurations in which the shape, texture, or coating of a surface 614or a surface 618 is compatible with, or which promotes, TIR;configurations in which the difference between the index of refractionof the material making up a controlled thickness area 613 and that ofthe material or space in contact with a surface 614 that forms aboundary of a controlled thickness area 613 is compatible with, or whichpromotes, TIR; configurations in which the difference between the indexof refraction of the material making up a support structure 604 and thatof the material or space in contact with a surface 618 that forms aboundary of a support structure 604 is compatible with, or whichpromotes, TIR; and other configurations and designs. In order to enhancethe TIR, the first material, within which the light is to be(internally) reflected should have a higher index than that of thesecond material into which the light would pass if it were notinternally reflected; since this second material is usually air, with anindex of refraction near 1, this is not usually difficult to ensure. Theangle of incidence must be greater than the critical angle in order toprovide TIR. For example, referring to embodiments shown in FIG. 8, thematerials making up controlled thickness area 613 and structures 604(e.g., the regions outside surfaces 614 and 618) should have an index ofrefraction that is greater than that of air. In embodiments where TIR isdesired within a layer 800, the material of the layer 800 should have alower index of refraction than that of controlled thickness area 613 toensure TIR occurs at the walls illustrated in FIGS. 8A, 8B, and 8D. Inalternative embodiments, the material of a layer 800 may have an indexof refraction that is higher than the index of refraction of thematerial of controlled thickness area 613, which will create TIR at thatboundary (between a layer 800 and a controlled thickness area 613)effective that the angles and materials may be adjusted so as tooptimize the trans-illumination component of light directed at a samplein an analysis area 608.

In embodiments, a surface 614 or 618 may be coated or treated so as toaffect or reduce reflectance (whether PIR or TIR) at the surface. Inembodiments, a surface 614 or 618 may be coated or treated so as toreduce light leakage out of the surface. For example, even where asurface 614 or 618 is compatible with, or enhances the amount of, TIR,some light may also be transmitted or refracted out of the surface 614or 618. A light-absorbing coating or material may be placed or appliedto such a surface 614 or 618, or to a portion or portions thereof, inorder to reduce the amount of stray light leaking from a cuvette 600.Such a light-absorbing coating may be, for example, a dye, an ink, apaint, a surface treatment, a black or colored tape, or other coating orsurface treatment. In embodiments, a black or other light-absorbingsolid material may be placed against or adjacent to a surface 614 or 618to provide an optically absorbent surface.

Optionally, in some embodiments, a cuvette 600 may be configured so asnot to provide TIR of light (or to provide only insignificant amounts ofTIR), or so as not to provide PIR (or only insignificant amounts ofPIR), from a portion, or portions, of the cuvette. For example, in someembodiments, a cuvette 600 may be configured so as not to provide TIR orPIR of light (or to provide only insignificant amounts of TIR or PIR)from the support structures 604. Optionally, in some embodiments, acuvette 600 may be configured so as not to provide TIR or PIR of light(or to provide only insignificant amounts of TIR or PIR) from a surface618. Configurations which do not provide TIR or PIR, or which provideonly insignificant amounts of TIR or PIR, include, without limitation:configurations in which the dimensions of controlled thickness area 613are incompatible with, or do not promote, TIR or PIR; configurations inwhich the angle or angles of a surface 614 or a surface 618 (e.g., withrespect to incident light) are incompatible with, or do not promote, TIRor PIR; configurations in which the shape, texture, or coating of asurface 614 or a surface 618 is incompatible with, or does not promote,TIR or PIR; configurations in which the difference between the index ofrefraction of the material making up a controlled thickness area 613 andthat of the material or space in contact with a surface 614 that forms aboundary of a controlled thickness area 613 is incompatible with, ordoes not promote, TIR or PIR; configurations in which the differencebetween the index of refraction of the material making up a supportstructure 604 and that of the material or space in contact with asurface 618 that forms a boundary of a support structure 604 isincompatible with, or does not promote, TIR or PIR; and otherconfigurations and designs.

Optionally, in some embodiments a reflective material may be placed at,or attached to, a surface 614 or a surface 618. Such a reflectivematerial may be, for example, a metal such as silver, or gold, oraluminum; may be a dielectric, such as magnesium or calcium fluoride, orother salt or metal oxide; or other reflective material. Typically, sucha reflective coating may be very thin (e.g., may be less than about 0.1micron, or may be up to about 100 microns thick). Optionally, areflective material (e.g., a reflective coating) may be placed at, orattached to, only surface 614. Optionally, a reflective material may beplaced at, or attached to, only surface 618. Optionally, surface 618 maybe treated to be black so as to be light absorbing. In otherembodiments, a surface 614 may be treated to be black so as to be lightabsorbing. Some embodiments may select the width of the controlledthickness area 613 to be wider than the analysis area 608. For someembodiments using laser illumination, the layer 800 may be removed or belight transmitting as the laser illumination is sufficiently focused soas not to require blackout between analysis areas 608.

By way of example and not limitation, the use of PIR, TIR, or both, canalso enable light traveling along path 820 from adjacent areas to bedirected into the analysis area 608. As shown in FIGS. 8A, 8B, and 8D,light traveling along path 820 is reflected towards analysis area 608,and light traveling along path 825 undergoes multiple reflections as ittravels within cuvette 600 and ultimately to analysis area 608. Asshown, light traveling along path 820 in FIG. 8B undergoes multiplereflections as it travels within cuvette 600 and ultimately to analysisarea 608. As illustrated in FIG. 8B, such reflections may be PIR or maybe TIR. Under traditional terminology, the illumination shown in FIG. 8Aby light traveling along paths 820 and 825, and the illumination shownin FIG. 8B by light traveling along path 820, is trans-illumination. Theillumination shown in FIGS. 8A and 8B by light traveling along paths 830shows light coming directly from the ringlight and not by way of TIR:this is epi-illumination. The combination of both types of lightcomponents from a light source located below the sample (or only oneside of the sample) allows for improved performance as compared tosources that can only provide one of those lighting components. This isparticularly useful for dark field microscopy.

One non-limiting example of the use of the embodiments shown in FIGS.8A-8D is dark field illumination to measure scatter properties of cellsin the sample. Dark field microscopy is an established method that hasbeen used mainly as a contrast-enhancing technique. In dark fieldmicroscopy, the image background is fully dark since only the lightscattered or reflected by the sample is imaged. Quantitative dark fieldmicroscopy has not been used to measure scatter properties of cells in amanner comparable to the use of traditional “side scatter” parameter inflow cytometers.

From the hardware perspective, illumination for dark field microscopy isdesired to be oblique, i.e. no rays of light from the illumination lightsource should be able to enter the objective without contacting thesample first. By way of example and not limitation, illumination shouldbe at a wavelength that does not excite any other fluorophores alreadypresent in the sample. Optionally, this illumination allows for the useof high numerical aperture (NA) lenses for imaging. By way of exampleand not limitation, for traditional lens sizes associated with opticalmicroscopes, the NA may be at least about 0.3. Optionally, the NA is atleast 0.4. Optionally, the NA is at least 0.5. Optionally, someembodiments may use oil immersion objective lenses to obtain a desiredNA, particularly when lens size is limited below a certain level.

Traditional methods for dark field illumination have usedtrans-illumination, where the sample is between the imaging lens anddark field light source. Thus, in this traditional arrangement, thedetection and illumination components are not on the same side of thesample. The traditional epi-illumination methods (where the imaginglens/objective and the light source are on the same side of the sample)require the use of specially manufactured objectives and typically donot allow the use of high NA objectives, thus limiting the capabilitiesof the whole system.

By contrast, at least some embodiments of dark field illuminationsystems described herein have the following attributes. In terms ofhardware, the scheme of the embodiments of FIGS. 8A-8D is “epi” in thatthe ringlight used for dark field illumination is on the same side ofthe sample as the objective. This can be desirable from thesystem-perspective, although alternative embodiments with light sourceson the other side may be used alone or in combination with theembodiments described herein. In one non-limiting example, the ringlightis designed such that the LEDs or lasers of the light source 654 are allin the same plane and have the same orientation (light sources in thesame horizontal plane and directing light upwards). Some embodiments mayhave light in the sample plane but directing light in a non-parallelmanner, such as but not limited to a cone-like manner. Some embodimentsmay have light in different planes but directing light in the sameorientation. Some embodiments may have light in different planes butdirecting light in a non-parallel manner, such as but not limited to acone-like manner. In some embodiments, the light is reflected by atoroidal mirror 652 to achieve oblique illumination of the sample.

In addition to the optical properties of the ringlight and the toroidalreflector, the optical properties of the cuvette 600 shown in theembodiments of FIGS. 8A-8D also significantly affects dark fieldillumination. In this embodiment, the cytometry cuvette 600 is designedsuch that light coming from the ringlight 650 falls directly on thesample; but in addition to this, light is also “reflected” on the samplefrom features of the cuvette so as to emulate “trans” illumination. Thisreflection can be by way of TIR or true reflection.

Note that any trans-illumination scheme allows one to measure forwardscattered light from a sample whereas an epi-scheme allows one tomeasure only the back-scattered light from the sample. Forward scatteredlight is generally two orders of magnitude greater in intensity than theback-scattered light. Thus, use of trans-illumination allows the use ofmuch lower illumination intensities and reduces harmful side-effects onthe sample.

As seen in the embodiment of FIG. 8A, the ringlight 650 (or other sourceof illumination) and cuvette 600 provide a system that can be tuned suchthat the intensities of trans and epi-illumination are adjusted forimproved performance over traditional epi-illumination. Similarly, theringlight 650 (or other illumination source) and cuvette 600 provide asystem in the embodiment of FIG. 8B that can be tuned such that theintensities of trans and epi-illumination are adjusted for improvedperformance over traditional epi-illumination. This tuning can beachieved by virtue of the materials chosen (e.g., for their opticalproperties) and design of cuvette geometry to control angles and extentof total internal reflection.

As shown in FIG. 8C, features 802 may alter the path of incident light,and so be used to enhance both trans-illumination and epi-illumination.As shown in FIG. 8D, the shape and configuration of surfaces 674, 676,and 678 may alter the path of incident light (e.g. transverseillumination), and so be used to provide or enhance trans-illumination,epi-illumination, or both.

FIG. 8E provides a schematic representation of transport of a cuvette600 from a sample preparation location to a sample observation locationnear an optical detector D. As indicated in the figure, a sample holder600 may be moved from one location to a location adjacent to, or on, adetector D. A detector D may include a stage configured to receive,hold, and position a cuvette 600. Sample may be added to the sampleholder via entry ports 602 (e.g., six entry ports 602 are shown in theexample shown in FIG. 8E), and may then be in a position for opticalobservation and measurement within an analysis area 608 (not shown, asinterior to the surfaces (e.g., of a support structure 604) of cuvette600 shown in FIG. 8E. Sample that is held within an analysis area 608may be illuminated, and may be detected by a detector D. In embodiments,a detector D may be configured to make qualitative observations orimages, and in embodiments a detector D may be configured to makequantitative observations or images.

A detector D as shown in FIG. 8E may comprise, or be part of, acytometry unit or cytometry module. Such a cytometry unit or cytometrymodule may comprise an independent unit or module for sample analysis.In embodiments, other analysis capabilities and devices may be includedin a detector D, or may be housed together with, or may be configuredfor use in conjunction with, a detector D. In embodiments, systems forsample analysis as disclosed herein may comprise such a cytometry unitor cytometry module, e.g., comprising a detector D used to analyze asample in a cuvette 600. In embodiments, systems for sample analysis asdisclosed herein may comprise such a cytometry unit or cytometry moduleand other units or modules which provide other analysis capabilities anddevices in addition to that of a detector D used to analyze a sample ina cuvette 600. In such systems, such other units or modules may behoused together with, or may be configured for use in conjunction with,a detector D. Such other analysis capabilities and devices may beapplied to a sample; for example, such analysis capabilities and devicesmay be used to analyze the sample or portion of a sample that is presentin a cuvette 600. In embodiments, such analysis capabilities and devicesmay be used to analyze a different portion of the sample present in acuvette 600 (e.g., a sample may be divided into two or more aliquots,where one aliquot is placed in a cuvette 600 for cytometric analysis,and one or more other aliquots are analyzed by other devices housed in,or near, or operated in conjunction with a cytometry unit or cytometrymodule. Thus, for example, independent of the analysis performed by sucha cytometry module, a sample (or portion thereof) may be measured oranalyzed in a chemical analysis unit, or in a nucleic acid analysisunit, or in a protein analysis unit (e.g., a unit using antibodies orother specifically binding molecules to analyze a sample), or other suchunit or combination of units and capabilities. Such analysis may includeanalysis for small molecules and elements present in a sample (e.g., bya general chemistry unit); analysis for nucleic acid molecules presentin a sample (e.g., by a nucleic acid unit); analysis for proteins orantibody-reactive antigens present in a sample (e.g., by anenzyme-linked immunosorptive assay (ELISA) unit); or combinations ofthese. In addition, systems as illustrated in FIG. 8E and as discussedherein may include a controller to control and schedule operations inone or more of the units or modules.

FIG. 8F provides a further, detailed schematic representation of systemincluding a transport mechanism for transporting a cuvette from a samplepreparation location to a sample observation location near an opticaldetector D. A system such as a system of the embodiment shown in FIG. 8Fmay include multiple sample analysis modules, which may be configured towork independently, or, in embodiments, may be configured to worktogether. The system shown in FIG. 8F includes a single cytometry unit707, with a detector D; in embodiments, samples (or portions thereof)analyzed in any or all of the analysis modules 701, 702,703,704, 705,and 706 may be transported to cytometry module 707, for observation andmeasurement by detector D. Independent of the analysis performed bycytometry module 707, a sample (or portion thereof) may be measured oranalyzed in a chemical analysis unit 715. Such analysis in a chemicalanalysis unit 715 may include analysis for small molecules and elementspresent in a sample (e.g., by a general chemistry unit); analysis fornucleic acid molecules present in a sample (e.g., by a nucleic acidunit); analysis for proteins or antibody-reactive antigens present in asample (e.g., by an ELISA assay unit); or combinations of these.

Systems as illustrated in FIG. 8F may include a controller to controland schedule operations in one or more of the modules 701-707. Samplesmay be loaded onto sample holders or other elements for analysis insystems as illustrated in the example shown in FIG. 8E. Such systems,and modules of such systems, include, e.g., sample handling systems 708;pipettes for obtaining, moving, and aliquotting samples, includingsuction-type pipettes 711 and positive displacement pipettes 712;centrifuges 713; spectrophotometers 714; chemical analysis units 715;photomultiplier tubes (PMTs) 716; cartridges 717 for holding disposablesupplies and tools, such as, e.g., pipette tips and other tips; andother elements. Modules and other elements may be supported by a rack709 or other support structure. Samples, disposables, tools, and otherelements may be transported within a module, and may be transportedbetween modules (e.g., between a module 701-706 and a cytometry module707).

FIGS. 8E and 8F show that the sample holder such as cuvette 600 may betransported from one location (such as where sample preparation mayoccur) and then to another location (such as to the detector D as seenin FIGS. 8E and 8F). The cuvette 600 does not release fluids into oronto the detector D, but instead is self-contained unit that keeps allof the sample therein. There may be one or more, two or more, or threeor more locations on or near to the detector D on which there istransparent surface on which the cuvette 600 or other sample holder canengage to provide a transparent interface for sample signal detection tooccur. Elements of FIG. 8F and further disclosure regarding suchelements and their uses can be found in U.S. patent application Ser. No.13/769,779, which is hereby fully incorporated by reference herein.

Dark Field

At least some embodiments herein include a dark field illuminationsource and cuvette. The relevant features of the cuvette 600 relate todesigning the cuvette dimensions and optical materials and the geometryof the cuvette. The cuvette increases the extent of dark fieldillumination through reflection (e.g., through TIR, or PIR, or both). Inone embodiment, the system may simultaneously use trans dark field andepi dark field illumination of a sample.

In some embodiments disclosed herein, the cuvette 600 combined with thelight source 650 enables trans and epi-illumination using a physicalsystem in the epi configuration (i.e., with the light source and theobjective on the same side of sample). The basic cuvette is designed tocontain the biological sample and present it for visualization. Inembodiments, the cover portion 612 may have a specific design. It isknown that different materials may have different indices of refraction;material having a desired index of refraction may be selected for use infabricating a cover portion 612, or a base support 620, or otherelements and components of a cuvette 600 and associated elements andcomponents. For example, in some embodiments, a cover portion 612 or abase support 620 may be made of glass. For example, in some embodiments,a cover portion 612 or a base support 620 may be made of quartz. Forexample, in some embodiments, a cover portion 612 or a base support 620may be made of an acrylic, or a clear polymer (e.g., a cyclo-olefin, apolycarbonate, a polystyrene, a polyethylene, a polyurethane, apolyvinyl chloride, or other polymer or co-polymer), or othertransparent material.

One can design the material of the top cover portion 612 to facilitateillumination and image collection. In embodiments, to illuminate asample, the light source 650 may be a ringlight 650 (i.e., may becircular), may have light sources 654 position in a discrete orcontinuous pattern, and may use a curved reflector 652 to direct lightto the sample.

In dark field microscopy, the sample is illuminated by oblique rays. Indark field microscopy, the light going into the microscope optics islight scattered by the sample, allowing the measurement of the scatterproperties of cells, particles, and other objects and structures in thesample. If no cells, particles, structures, or other objects are presentin the sample, then the dark field image is black.

In the present non-limiting example, the reflector 652 and LED 654 ofthe ringlight 650 are designed to reflect light so that a minimumfraction of light goes directly back into the objective as non-specificbackground. The system is designed to direct light by TIR at cuvettesurfaces back into the analysis area 608. Light reflected from asurface, whether by TIR or other reflection, is thus directed toilluminate a sample in the analysis area 608. The cells, particles, andstructures in the sample in analysis area 608 receive light directlyfrom the ringlight from underneath the cell (i.e., viaepi-illumination). In addition, as disclosed herein, light coming fromthe top surfaces (reflected) is also directed to the analysis area 608(i.e., via trans-illumination).

Thus, according to the systems and methods disclosed herein, with theringlight 650 in the same position, light may be directed to illuminateanalysis area 608 from two directions (both epi-illumination andtrans-illumination) from a single ringlight source. In embodiments, thisillumination is all oblique illumination. One can control the relativestrengths of the two light components by design of the cuvette andmaterial used for the cuvette.

This dark field illumination is different from conventional dark field.For example, in embodiments disclosed herein, dark field illumination isprovided by light reflected at a cuvette surface by TIR. By way ofnon-limiting example, in embodiments, a system as disclosed herein mayuse a reflective layer on the backside of certain surfaces of the coverportion 612 to reflect all of the light. By way of non-limiting example,in embodiments, a system as disclosed herein may use a reflective layeron the backside of certain surfaces of a cuvette 600 to reflect all ofthe light. Some embodiments may use a full or selectively reflectivebackground.

For example, in embodiments, it is desirable to direct the light at anoblique angle, which keeps illumination dark field. In some embodimentslight sources 654 may direct light at an angle, and thus may not requireor may not use the reflector 652. The reflector 652 may improvemanufacturability of the light source 654 since all lights are in thesame plane, directed in the same direction. Optionally, the angled lightsources 654 may also be used in place of or in combination with areflector.

It should be understood that even though the light intensity of atrans-illumination component of illumination may be, e.g., 10 timesweaker than a corresponding epi-illumination component, the intensity oflight scattered from the cells or other objects in the sample due totrans-illumination may be 200 times stronger. That is, where scatterfrom an amount of epi-illumination is compared to scatter from the sameamount of trans-illumination, the intensity of light scattered due totrans-illumination may be 200 times stronger than the light scattered byepi-illumination of cells or other objects in the sample. Thus, a smallamount of trans-illumination can significantly enhance the light scatterfrom cells.

With epi-illumination alone, light collected by an objective is onlythat light reflected from a sample. However, diffraction is asubstantial component of scatter and the use of trans-illuminationprovides for some amount diffraction (e.g., light diffracted by thesample). However, the light collected from epi-illumination does notinclude light diffracted by the sample (without reflection of the lightback towards the light source following diffraction). Thus, when usingtrans and epi-illumination there are reflective, refractive, anddiffractive components to the light collected by an objective.Traditional methods use all trans dark field illumination which takes asignificant amount of space to configure, due to the placement ofoptical components on both sides of the sample. In contrast, systems andmethods as disclosed herein provide both epi-illumination andtrans-illumination using optical elements configured forepi-illumination alone. The embodiments disclosed herein may obtain thespace savings of an epi-illumination configuration while providing thebenefits of both epi- and trans-illumination of the sample.

Designing the sample holder and the light source together can enable anepi-illumination configuration to increase the amount oftrans-illumination of the sample, and in particular may provide uniformtrans-illumination. Some embodiments may use mirrored surfaces. Someembodiments use TIR, which can be tuned to create the desiredtrans-illumination, including trans-illumination that is uniform and atoblique angles into the analysis area 608 for dark field illumination ofthe sample. A cuvette 600 may be configured so as to providetrans-illumination of an analysis area 608 solely from a light source inan epi-illumination configuration using reflection, e.g., using TIR orPIR, or both. In one non-limiting example, a thicker cover portion 612allows the light undergoing TIR (or PIR, or both) to reflect back intothe target area 608. Additionally, the systems and methods disclosedherein not only provide light that, due to TIR (or PIR, or both), comesback into an analysis area 608. but light that comes back into ananalysis area 608 uniformly. The embodiments of FIGS. 8A, 8B, and 8Dhave certain surfaces at certain angles, have certain black surface(s),and certain reflective surface(s) so that the light comes back uniformlyto an analysis area 608 effective to provide uniform trans-illuminationof a sample in an analysis area 608. Optionally, one could put a fullyreflective surface on a top (such as but not limited to a flat coverportion 612 as shown in FIGS. 7A and 7B, and optionally over selectareas of a top of an area 613 of FIGS. 8A, 8B, and 8C). In contrast,light traveling within traditional hardware may undergo some reflection,including possibly some TIR (or PIR, or both), but the light may notcome back into the area 608.

By way of non-limiting example, embodiments disclosed herein take animaging based platform and instead of using a high complication, highcost system which may for example have 16 laser light sources, thepresent embodiment leverages a more integrated detection system to beable to image and identify the differentials of cells and types in asample.

In one non-limiting example, the combination of all these differenttypes of information is useful and effective to achieve the desiredgoals of the analysis. This may include quantitative measurements orqualitative measurements linked to quantitative measurements, or imageslinked to quantitative measurements. The methods and systems disclosedherein provide different channels of fluorescence where each channel mayhave one or more specific molecular markers targeted (i.e., quantitativeinformation). The methods and systems disclosed herein may include, andmay be used with, microscopy, embodiments herein may provide the abilityto observe and measure the background that staining forms inside thecell (e.g., whether it is in the cytoplasm, is it concentrated on thesurface, in the nucleus, or elsewhere) that can link image orqualitative information that is generated to quantitative measurementsthat are generated. In this manner, the linkage of the original imagesthat created the quantitative results are available for further analysisif it turns out that the quantitative measurements trigger alarms ormeet thresholds the suggest further analysis is desired. Embodimentsherein can interrogate background images and information that stainingcreates in a cell in a sample within an analysis area 608. Such imagesand information allow the determination of whether or not the stainingis in the cell, e.g., in the cytoplasm, in the nucleus, in the membrane,or other organelle or cellular location.

In some embodiments of the methods and systems disclosed herein,combinations of the quantitative scatter properties of the cell, theshape of the cell, or the size of the cell may be observed and measured,and used to identify or characterize a sample. In some embodiments ofthe methods and systems disclosed herein, the physical properties,optical properties, and bio/biochemical properties of a sample orportion thereof may be observed and may be measured all in the samedevice at the same time. All such measurements and observations can becombined in a programmable processor or other processing system to linkthe various types of information to achieve the goals of the assays(e.g., to achieve a clinical goal of the assays).

Although traditional devices may be suitable for one or the other kindof observation or measurement, they are not suitable for bothepi-illumination and trans-illumination from a single light source;there is also no linkage between such different types of information.For example, in some embodiments disclosed herein, where imageinformation that generated the quantitative measurements is retrievable,the systems and method may be used for tissue morphology measurements.Optionally, the system can be applied to pap smear, which is moresimilar to traditional cytology. It can be extended to anything doneusing traditional microscopy. In urine, at least some of the presentembodiments can look at and analyze crystals and not just cells. One canlook at crystals of inorganic salts and chemicals from urine samplesthat had created certain quantitative readings on one portion of agraph. In addition, one can look at and analyze cells and particlespresent in blood, including analysis of different types and populationsof blood cells, such as but not limited what may be seen in FIG. 1Awhere different regions of data are circled. Image information forcertain data regions can be retrieved to further analyze the underlyingcell images that created the measurements plotted on the graph or chart.

Some embodiments herein combine the imaging features with the pathologyfeatures. For example, tissue preparation may occur inside a device orsystem configured to include the optical elements disclosed herein (asystem may be, or include, for example, a module or multiple modulesconfigures for optical and other analysis of a sample), and suchprepared material can be imaged in this platform. Then the image oranalysis may be sent to servers to do image analysis, to do diagnosis,or to perform digital pathology effective to aid or enable a pathologistto analyze a sample.

Embodiments of methods, systems and devices as disclosed herein,including, e.g., systems and devices illustrated in FIGS. 8C and 8D,provide a wide range of cytometry capabilities which may be appliedtogether to analyze a sample. Such cytometry capabilities includecytometric imaging such as is typically confined to microscopy; suchmicroscopic imaging and image analysis of biological samples is providedby the devices, systems, and methods disclosed herein. In addition, thesystems and devices as disclosed herein are configured to providespectrophotometric analysis of biological samples. Such image analysisincludes dark field, brightfield, and other image analysis. Novel andimproved methods for applying both epi-illumination andtrans-illumination from a single light source are disclosed, which allowmore sensitive and accurate images and analysis of blood samples. Inconjunction with the methods disclosed herein, separate measurementsregarding RBCs, WBCs, and sub-categories of these may be obtained. Imageand spectrophotometric analysis as disclosed herein may be used toidentify and quantify different populations of WBCs useful for thecharacterization of a blood sample and for the diagnosis of manyclinical conditions. Devices and systems as disclosed herein may be usedto provide clinical reports which include general chemical analysisinformation, nucleic acid-based analysis information, antibody- (orprotein or epitope)-based analysis information, spectrophotometricanalysis information, and in addition provide images of the cells andsamples analyzed. The ability to produce such information and to providesuch reports, including images as well as other clinical information, isbelieved to provide novel and unexpected capabilities and results.

In addition, this information, and these reports, may be produced in ashort amount of time (e.g., in less than an hour, or less than 50minutes, or less than 40 minutes, or less than 30 minutes, or othershort amount of time). In addition, this information, and these reports,may be produced from small samples, e.g., small samples of blood orurine. Such small samples may have sizes of no more than about 500 μL,or less than about 250 μL, or less than about 150 μL, or less than about100 μL, or less than about 75 μL, or less than about 50 μL, or less thanabout 40 μL, or less than about 20 μL, or less than about 10 μL, orother small volume. In embodiments where a sample is a blood sample,such small sample may be collected from a finger-stick. Typically, onlya small amount of blood is collected from a finger-stick (e.g., theamount of blood may be about 250 μL or less, or about 200 μL or less, orabout 150 μL or less, or about 100 μL or less, or about 50 μL or less,or about 25 μL or less, or other small amount).

Clinical reports which include cytometric information and images, asdisclosed herein (including images, scatter plots, and other optical andimaging information), and which also include general chemical analysisinformation, nucleic acid-based analysis information, antibody- (orprotein or epitope)-based analysis information, and spectrophotometricanalysis information, are believed to provide broad and clinically richinformation useful for the diagnosis and characterization of manyclinical conditions, and to provide advantages over the art. Suchreports may be prepared rapidly at a point of service (or point of care)location, and may be rapidly communicated (e.g, electronically bywireless, land-line, optical fiber, or other communication link) to apathologist or other clinical expert for analysis and interpretation.Such expert analysis and interpretation may then in turn be rapidlycommunicated (e.g, electronically by wireless, land-line, optical fiber,or other communication link) to a clinician caring for the subject, orback to the point of service (or point of care) location, or both, forrapid feedback. Such rapid feedback enables timely treatment, ifnecessary, or prevents unnecessary treatment, by providing informationand analysis based on samples which may be acquired, may be analyzed, orboth, at a point of service or point of care location. Such rapidanalysis, reporting, and feedback provides advantages overtime-consuming methods, and, by allowing timely treatment and byavoiding unnecessary treatment, may provide more effective, moreefficient, and less costly clinical services and treatment. Such moretime-consuming methods which may be obviated by the devices, systems andmethods disclosed herein include, but are not limited to: delay andinconvenience due to a subject being required to travel to a laboratoryor clinic remote from the subject's home, and remote from the clinicianentrusted with the care of the subject; delays and possible sampledegradation due to transport of a sample from a collection location to alocation where the sample may be analyzed; delays due to transmission ofthe results of such analysis to a pathologist or other expert; delaysdue to transmission of an expert opinion to the subject's clinician;delays in transmission of clinician diagnosis and treatment of thesubject following transmission of an expert opinion to the clinician.These delays, inconveniences, and possible sample degradation may bereduced or eliminated by use of the methods, devices, and systemsdisclosed herein.

Embodiments of systems and devices as illustrated in FIGS. 6A, 6B, 7,8A, 8B, 8C, and 8D, and other figures and as disclosed herein, providecytometry capabilities in a compact format, including in compact formatsfor use with one or more other sample analysis capabilities. Applicantsdisclose herein novel devices and systems which include the novelcytometry capabilities as disclosed herein in devices and systems alongwith other sample analysis capabilities. For example, Applicantsdisclose herein devices and systems which provide novel cytometrycapabilities as disclosed herein in conjunction with devices and systemsfor sample analysis by a general chemistry unit; in conjunction withdevices and systems for sample analysis by a nucleic acid analysis unit;in conjunction with devices and systems for sample analysis usingantibody assays (e.g., ELISA) unit); and combinations of these. Thus, asample processing device as disclosed herein may be configured toperform a plurality of assays on a sample. Such a sample may be a smallsample.

In embodiments, all sample assay actions or steps are performed on asingle sample. In embodiments, all sample assay actions or steps areperformed by a single device or system and may be performed within ahousing of a single device. Such systems and devices includingcytometry, particularly cytometry which provides image analysis as wellas spectrophotometric or other optical analysis in a single unit, arebelieved to be novel and unexpected. Providing systems and devicesincluding cytometry, particularly cytometry which provides imageanalysis as well as spectrophotometric or other optical analysis in asingle unit, is believed to provide advantages previously unavailable inthe art.

Embodiments of systems and devices as illustrated in FIGS. 6A, 6B, 7,8A, 8B, 8C, and 8D, and other figures and as disclosed herein, providecytometry capabilities in a portable format, where such devices andsystems may be housed in enclosures small enough for easy transport fromone location to another. For example, such devices and systems may bereadily transported for use at a point of care location (e.g., adoctor's office, a clinic, a hospital, a clinical laboratory, or otherlocation). For example, such devices and systems may be readilytransported for use at a point of service location (in addition to suchpoints of care locations discussed above, e.g., a pharmacy, asupermarket, or other retail or service location). A point of servicelocation may include, for example, any location where a subject mayreceive a service (e.g. testing, monitoring, treatment, diagnosis,guidance, sample collection, ID verification, medical services,non-medical services, etc.). Point of service locations include, withoutlimitation, a subject's home, a subject's business, the location of ahealthcare provider (e.g., doctor), hospitals, emergency rooms,operating rooms, clinics, health care professionals' offices,laboratories, retailers [e.g. pharmacies (e.g., retail pharmacy,clinical pharmacy, hospital pharmacy), drugstores, supermarkets,grocers, etc.], transportation vehicles (e.g. car, boat, truck, bus,airplane, motorcycle, ambulance, mobile unit, fire engine/truck,emergency vehicle, law enforcement vehicle, police car, or other vehicleconfigured to transport a subject from one point to another, etc.),traveling medical care units, mobile units, schools, day-care centers,security screening locations, combat locations, health assisted livingresidences, government offices, office buildings, tents, bodily fluidsample acquisition sites (e.g. blood collection centers), sites at ornear an entrance to a location that a subject may wish to access, siteson or near a device that a subject may wish to access (e.g., thelocation of a computer if the subject wishes to access the computer), alocation where a sample processing device receives a sample, or anyother point of service location described elsewhere herein.

Esoteric Cytometry and Specialty Cytometry Markers

Many traditional advanced or esoteric cytometric assays require atraditional system to measure a large number of markers on cells;typically, these markers are measured simultaneously. The generalapproach in the field has been tied to high capability instrumentsincluding, for example, six or more lasers and 18 different PMT tubes tomeasure all of these markers simultaneously. However, in many clinicalsettings, simultaneous measurements of multiple markers are notrequired. In many clinical requirements, for example, one is interestedin seeing how many cells are positive for one marker, or how many arepositive for a combination of two or three markers, or other suchcombination of a few markers. Some embodiments herein provide formultiple combinations of staining schemes where one may have a set of,for example, 10 markers, where one can combine them in sets of 3-4 or5-6 markers where one can combine them such that even if combining twomarkers in the same color, some embodiments of the present system cande-convolute the images and information in order to determine whichsignal came from which marker. This allows some embodiments of thepresent system to reduce the hardware requirements in terms of thenumber of light sources, the number of channels used for sampleanalysis, and other simplifications and efficiencies. Thus, usingsubsets of a number of markers, or using or measuring markers innon-simultaneous manner in a pre-determined pairing can be useful toenable esoteric cytometry. For example, some markers may be consideredto be “gating” markers; such markers are measured first, and if theresults of such initial measurements are negative (e.g., the markers arenot present, or are present only in low amounts, in a sample), thenmeasurements using other, follow-on markers may not be needed. Inembodiments such non-simultaneous methods and systems may reduce thesample volume required for analysis, and may reduce the amounts ofmarkers needed for analysis (e.g., if a follow-on marker is typicallyused in only a small fraction of samples analyzed).

It should be understood that the use of imaging for cytometric analysesof samples, such as blood or urine samples, enables one to obtain anactual cell count, and so may be more accurate than traditionalcytometry methods which do not include such measurements. Imaging ofsamples, including imaging of cells (and particles or structures) in asample can actually be more accurate than other methods, such astraditional flow cytometry. For example, traditional flow cytometrygating does not allow for actual counts. The gating in flow cytometry issubjective and thus this can vary from system to system. In addition,traditional flow cytometry does not provide images of cells in a sample.

Some embodiments herein may also gate, but the gating is basedalgorithmically based on various factors including but not limited topatient health. Classification means is trained on a population ofpatients knowing if they are healthy or diseased. Some embodiments herecan flag a patient that is abnormal and flagging it for review.Self-learning gating can determine if different gating is desired basedon information conveyed regarding the patient health. Thus, the gatingfor the sample for some embodiments disclosed herein is donealgorithmically, possibly with a programmable processor, and the gatingchanges based on patient health.

In embodiments of methods and systems for imaging, one may want tominimize the amount and complexity of hardware required, and one maywish to re-use some or all of the sample if possible, in order tominimize the sample volume required. Thus, the more capability one canextract from the imaging of a sample, the better in terms maximizing theinformation obtained from a sample, and where possible, from smalleramounts of sample. Thus, the more information one can get todifferentiate different cell types from a minimum number of pictures,the more one may minimize the sample volume required.

Optionally, in one non-limiting example, the cuvette for use in themicroscopy stage can be configured as follows (with reference to theembodiments and elements shown in FIGS. 7, 8A, and 8B). A middle channellayer comprises a core of thin plastic membrane 800 withpressure-sensitive-adhesive (psa) on both sides. One side adheres to thewindow-layer 606 and the other side to the molded-top-layer coverportion 612. The core is an extruded film that is black in color,primarily due to optical reasons of preventing light scatter and opticalcross-talk between the different liquid channels. The thickness of thecore membrane preferably is uniform along its length and width, and maybe formed, for example, from an extruded film of black PET or black HDPE(polyethylene). The psa sub-layers on both sides are preferably as thinas possible for preserving the tight and uniform dimensions of theoverall liquid channel (e.g., analysis area 608), yet are preferablythick enough to provide a good fluidic seal around the liquid channel.In embodiments, the psa adhesives useful for such sample holders areacrylic in nature and have high adhesion strength for low-surface-energyplastics. The liquid channels, ports and other alignment features on themiddle layer may be fabricated using laser-cutting or die-cuttingprocesses. In embodiments, heating of material to near, but not above,the melting point of the material may be used in the fabrication ofcuvettes, and cuvette chambers. In embodiments, diffusion bonding may beused in the fabrication of cuvettes, and cuvette chambers (e.g., cuvettecomponents may be heated to their materials' glass transitiontemperature, allowing or enhancing diffusion of material betweenpreviously separate components of a cuvette); for example, acrylic toacrylic bonds may be made using diffusion bonding. In embodiments,ultrasonic welding may be used in the fabrication of cuvettes, andcuvette chambers. For example, bonding methods including, but notlimited to use of heating, use of adhesives, use of diffusion bonding,use of ultrasonic welding, and other suitable techniques and methods,may be used to bond a support structure to a cover portion of a cuvette(e.g., a support structure 606 to a cover portion 612 of FIGS. 7A and7B). Sonically welding cuvettes, such as but not limited toultrasonically welding them, may involve make multiple layers of thecuvette and putting them together, rather than molding or usingadhesives for the multiple layers. In embodiments, various techniquesmay be combined for manufacturing of the cuvette such as but not limitedto ultrasonically welding certain layers while using adhesives or otherbonding techniques on other layers. Optionally, some embodiments may useone technique to bond perimeter portions of the cuvette while anothertechnique may be used to bond structures or layers that will come incontact with sample or liquids when the cuvette is in use.

A channel in a cuvette may have an entry port (e.g., an entry port 602as shown in FIG. 6A) for filling, and may have two or more entry ports602 for filling. An entry port 602 may have any shape or configurationsuitable for transfer of sample into the interior of the channel. Inembodiments, an entry port may have a round, or oval, or other shapesuitable to allow a pipette (e.g., a pipette with a conical or similarlytapered end-portion) to transfer a fluid sample to and into a channel.For example, a round entry port may be suitable to accept a tip of aconical pipette where the pipette is oriented substantiallyperpendicular to the plane of the entry port. For example, an oval entryport may be suitable to accept a tip of a conical pipette where thepipette is oriented at an angle from the perpendicular to the plane ofthe entry port. For example, an entry port may be configured to allowspace for an end-portion of a pipette (e.g., a pipette tip) to bepositioned over the entry port effective that fluid exiting the pipettetip falls or otherwise flows into the entry port; in embodiments, aspace may remain between at least a portion of the entry port and atleast a portion of the pipette tip. In embodiments, an entry port may beconfigured to contact or otherwise engage with at least one portion ofthe liquid dispensing tip such as but not limited to an end-portion of apipette (e.g., a pipette tip) so as to form a seal between theend-portion of the pipette and the walls of that entry port. Inembodiments, an entry port may have an internal taper (e.g., thediameter or other cross-sectional length of the outer-most portion of anentry port may differ from the diameter or other cross-sectional lengthof the inner-most portion of that entry port). In embodiments of anentry port with an internal taper, the inner diameter or othercross-sectional length of the entry port may be smaller than thediameter or other cross-sectional length of the outer-most portion ofthat entry port, effective to complement the taper of a pipette tip(e.g., a conical pipette tip) positioned in the entry port. Inembodiments, a pipette tip may engage with an entry port effective toprevent fluid (e.g., sample) delivered by the pipette from flowing outof the channel via the entry port. Optionally, the port in the cuvettemay be sized or otherwise designed to form a seal against at least someportion of the pipette tip. Optionally, the material may be ahydrophobic material so that liquid only enters the cuvette whensufficient force dispenses the liquid from the tip, and not primarilydue to any hydrophilic force.

In embodiments, a channel in a cuvette may have a vent effective toallow air or other gas to flow (e.g., to exit) aiding filling of achannel with sample (e.g., a fluid sample such as blood, or plasma, orother fluid). In embodiments, an entry port may serve as a vent, or, inembodiments, a channel may have a vent separate from, and in additionto, an entry port. In embodiments, a vent may comprise a porous membraneconfigured to allow passage of air or gas yet to reduce or preventevaporation of liquid from the channel (e.g., from a sample within thechannel). Such a vent may be covered with a porous membrane, or mayinclude a porous membrane at or near the opening of the vent. Porousmembranes made with hydrophobic materials may be more effective tomitigate evaporation from a sample than porous membranes made withhydrophilic materials. Such a porous membrane may be made with, e.g., acyclo-olefin polymer such as Zeonex® or Zeonor® (Zeon Chemicals,Louisville, Ky., USA); polyethylene (PE); polyvinylidene fluoride(PVDF); combinations of PE and PVDF such as Porex® (Porex Corporation,Fairburn, Ga., USA); or with other porous materials and combinations ofmaterials.

A channel in a cuvette may be filled, for example, by providing sampleto an entry port of a channel. It will be understood that by “filling achannel” both complete, and partial, filling of the channel is meant;thus “filling a channel” as used herein refers to filling a channel, orportion of a channel, whether the channel becomes completely or onlypartially filled. A fluid sample may be provided to a channel by gravityflow into the channel, e.g., via an open entry port. A fluid sample maybe drawn into a channel by capillary action; for example, contact of adrop or portion of sample provided by a pipette tip with a wall of achannel via an entry port may initiate and provide capillary flow ofsample into a channel. Such a capillary means of filling a channel ismore effective, and more readily accomplished, where the walls of thechannel, or at least the interior surfaces of the channel, comprisehydrophilic materials or coatings. In embodiments, filling a channel maybe accomplished using pressure, where fluid is forced into the channelby application of force (e.g., by hydraulic or air pressure, which maybe supplied by a piston, a pump, compressed gas, osmotic pressure, orother means). Where a channel is filled by pressure, hydrophobicmaterials may be used to form, or coat, the interior walls of thechannel. Such hydrophobic materials (e.g., including acrylics, olefins,cyclo-olefins, and other polymers and plastics) may provide betteroptical properties than other (e.g., than some hydrophilic) materials.Where a channel is to be filled using pressure, a tight seal between apipette (used to deliver the fluid, e.g., the sample) and the entry portof the channel may be preferred. Where a channel is to be filled usingpressure, a vent (or vents) configured to allow exit of gas (e.g., air)or liquid previously occupying some or all of the channel interior maybe provided. Use of pressure to fill a channel allows for control of therate and volume of fluid delivered; such rate and volume control may begreater than the control of rate and volume accomplished when usingcapillary or gravity flow to fill a channel.

In embodiment as disclosed herein, magnetic elements may be incorporatedinto the cuvette (such as but not limited to magnetic pucks or discs, ormetal pucks or discs that may be held by a magnet). For example, suchmagnetic elements may be included in, or may comprise, the molded toplayer of a sample holder or cuvette. Magnetic elements can be used tosimplify hardware used to transport the cuvette. For example, thehandling system can engage the magnetic features in the cuvette totransport it without having to add an additional sample handling device.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, different materials may be used to create differentreflective surfaces in the cuvette or other surfaces along a lightpathway in the optical system. Optionally, the reflective surface isselected so that the reflection is only diffusive. Optionally, thereflective surface is selected so that the reflection is only specular.Some embodiment may use a flat top illumination scheme as set forth inCoumans, F. A. W., van der Pol, E., & Terstappen, L. W. M. M. (2012),Flat-top illumination profile in an epifluorescence microscope by dualmicrolens arrays. Cytometry, 81A: 324-331. doi: 10.1002/cyto.a.22029,fully incorporated herein by reference for all purposes.

Optionally, some embodiments may have all channels having a bottomsurface in one plane, but due to different channel sizes, have topsurfaces in different planes. Optionally, some embodiments may havechannels in different vertical planes. Although most embodiments hereinshow imaging in a vertical top-down configuration, it should beunderstood that some embodiments may arrange channels in a verticallystacked configuration and image channels from the side. Some embodimentsmay use multiple cuvettes on an imaging platform. For example, althoughFIG. 8E shows a single cuvette thereon, it is possible to place multiplecuvettes onto the imaging platform for processing in sequential orsimultaneous manner. Although the cuvettes herein are typically shown asformed from transparent materials, some embodiments may form at leastsome portions of the cuvette from non-transparent material. This can beprovided to provide improved structural rigidity to portions of thecuvette and/or optionally, provide different light handling properties.Optionally, some embodiments may be used with a non-transparent carrierthat engages at least a portion of the cuvette and is moved with thecuvette to an imaging platform to facilitate handling and/or provide adesired optical effect.

Additionally, concentrations, amounts, and other numerical data may bepresented herein in a range format. It is to be understood that suchrange format is used merely for convenience and brevity and should beinterpreted flexibly to include not only the numerical values explicitlyrecited as the limits of the range, but also to include all theindividual numerical values or sub-ranges encompassed within that rangeas if each numerical value and sub-range is explicitly recited. Forexample, a size range of about 1 nm to about 200 nm should beinterpreted to include not only the explicitly recited limits of about 1nm and about 200 nm, but also to include individual sizes such as 2 nm,3 nm, 4 nm, and sub-ranges such as 10 nm to 50 nm, 20 nm to 100 nm, andother ranges.

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications mentioned herein are incorporated herein by referenceto disclose and describe the structures or methods in connection withwhich the publications are cited.

While the above is a complete description of the preferred embodiment ofthe present invention, it is possible to use various alternatives,modifications and equivalents. Therefore, the scope of the presentinvention should be determined not with reference to the abovedescription but should, instead, be determined with reference to theappended claims, along with their full scope of equivalents. Anyfeature, whether preferred or not, may be combined with any otherfeature, whether preferred or not. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means for.” It should be understood that as used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein and throughout theclaims that follow, the meaning of “in” includes “in” and “on” unlessthe context clearly dictates otherwise. As used herein, the term “or”may include “and/or”; thus, the meaning “or” includes both theconjunctive and disjunctive unless the context expressly dictatesotherwise.

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to facsimilereproduction of the patent documents and disclosures, as they appear inthe US Patent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever. The following notice shallapply: Copyright 2013 and 2014 Theranos, Inc.

1. A system for analyzing a sample, the system comprising: a sample holder comprising a sample chamber configured to hold said sample, at least a portion of said sample holder comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface; and an illumination source configured to provide light that illuminates and passes through said optically transmissive surface; wherein said sample holder is configured effective that said light from said illumination source simultaneously provides both epi-illumination and trans-illumination to a sample in the sample holder, where epi-illumination comprises light traveling from said illumination source to said sample without reflection at a surface of the optically transmissive material of the sample holder, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material.
 2. The system of claim 1, wherein the sample holder comprises a cuvette having an elonagated channel configured for holding a sample.
 3. The system of claim 1, wherein the sample holder comprises one or more optically non-transmissive surfaces.
 4. The system of claim 1, wherein said trans-illumination is provided at least in part by total internal reflection of light at a surface.
 5. The system of claim 2, wherein said trans-illumination is provided at least in part by total internal reflection of light within the cuvette.
 6. The system of claim 1, wherein the sample holder comprises two or more sample chambers for holding sample.
 7. The system of claim 2, wherein the cuvette has a rectangular horizontal, cross-sectional shape.
 8. The system of claim 2, wherein the cuvette has a circular horizontal, cross-sectional shape.
 9. The system of claim 2, wherein the cuvette has a saw tooth vertical cross-sectional shape.
 10. The system of claim 2, wherein the cuvette has a step-shaped vertical cross-sectional shape.
 11. The system of claim 1, wherein said sample holder is movable relative to said illumination source to a plurality of locations, wherein said optically transmissive surface of the sample holder may be illuminated by the illumination source at each of said locations.
 12. The system of claim 1, wherein said illumination source comprises a ringlight.
 13. The system of claim 12, wherein said ringlight is selected from a light emitting diode (LED)-based ringlight and a laser-based ringlight.
 14. The system of claim 1, further comprising a support structure comprising an optically transmissive surface shaped to engage an optically transmissive surface of the sample holder.
 15. The system of claim 1, further comprising a compression device configured to retain the sample holder in a desired location for illumination by the illumination source.
 16. The system of claim 1, further comprising a detector configured to image at least a portion of a channel in the sample holder.
 17. The system of claim 16, wherein said sample holder comprises an elongated channel configured to contain at least a portion of the sample, and wherein said detector is configured to image an entire elongated channel in the sample holder.
 18. The system of claim 16, wherein the sample holder is configured to hold the sample in a static, non-flowing manner during imaging.
 19. The system of claim 16, wherein during imaging, the sample holder is configured to hold one portion of the sample in a static, non-flowing manner and another portion in a flowing manner.
 20. The system of claim 16, wherein said illumination source is movable relative to the sample holder.
 21. The system of claim 1 wherein during imaging, the sample holder is configured to hold the sample in a flowing manner.
 22. The system of claim 16, wherein said sample holder further comprises a fluid circuit fully confined in the sample holder, and wherein the sample is located in said fluid circuit, effective that the sample remains separate from said detector.
 23. The system of claim 22, wherein said sample holder is movable relative to the detector.
 24. The system of claim 22, wherein said detector is movable relative to the sample holder.
 25. The system of claim 1, wherein said sample holder and said illumination source comprise at least part of an optical analysis unit, said system further comprising a clinical analysis unit configured to perform clinical analysis on said sample.
 26. The system of claim 25, wherein said system is configured to provide an aliquot of a single sample to each of said optical analysis unit and said clinical analysis unit, effective that said clinical analysis unit and said optical analysis unit may perform optical analysis and clinical analysis on portions of a sample at the same time.
 27. The system of claim 25, wherein said clinical analysis is selected from general chemical analysis, nucleic acid analysis, and enzyme-linked binding analysis.
 28. The system of claim 25, comprising a plurality of clinical analysis units, wherein each clinical analysis unit of said plurality of clinical analysis units is configured to provide a clinical analysis selected from general chemical analysis, nucleic acid analysis, and enzyme-linked binding analysis.
 29. A cuvette comprising a sample chamber configured to hold a sample, at least a portion of said cuvette comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface, wherein said optically transmissive surface and said reflective surface are configured effective that light passing through the optically transmissive surface simultaneously provides both epi-illumination and trans-illumination to said sample in the sample chamber, where epi-illumination comprises light traveling from an illumination source to the sample without reflection at a surface of the optically transmissive material, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material.
 30. The cuvette of claim 29, wherein the sample chamber comprises an elongated channel.
 31. The cuvette of claim 29, further comprising one or more optically non-transmissive surfaces.
 32. The cuvette of claim 29, wherein said trans-illumination is provided at least in part by partial internal reflection of light at a surface.
 33. The cuvette of claim 29, wherein said trans-illumination is provided at least in part by total internal reflection of light at a surface.
 34. The cuvette of claim 29, wherein the sample holder comprises two or more sample chambers for holding sample.
 35. The cuvette of claim 29, comprising a cross-sectional shape selected from rectangular horizontal cross-sectional shape and a circular horizontal cross-sectional shape.
 36. The cuvette of claim 29, comprising a cross-sectional shape selected from a saw tooth vertical cross-sectional shape and a step-shaped vertical cross-sectional shape. 37-40. (canceled)
 41. A method of identifying a cell in a sample containing a plurality of cells, comprising: (a) placing said sample in a sample holder comprising a sample chamber configured to hold the sample, at least a portion of said sample holder comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface, wherein said optically transmissive surface and said reflective surface are configured effective that light passing through the optically transmissive surface simultaneously provides both epi-illumination and trans-illumination to the sample in the sample chamber, where epi-illumination comprises light traveling from said illumination source to the sample without reflection at a surface of the optically transmissive material, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material; (b) illuminating said sample holder effective to simultaneously provide both epi-illumination and trans-illumination of the sample; and (c) identifying a cell in the sample.
 42. The method of claim 41, wherein said identifying comprises identifying said cell with a detector configured to image at least a portion of said sample chamber.
 43. The method of claim 42, wherein said sample chamber comprises an elongated channel.
 44. A method for the measurement of a component of interest in cells of a cellular population in a sample, comprising: a) obtaining a quantitative measurement of a marker present in cells of the cellular population in the sample; b) determining, with the aid of a computer, an approximate amount of cells in the cellular population present in the sample based on the measurement of part a); c) adding an amount of a cell marker to the sample, where the amount of said cell marker added is based on the results of part b), and wherein the cell marker binds specifically to the component of interest in cells of the cellular population and is configured to be readily detectable; d) assaying cells in the sample for marker bound to the component of interest; and e) determining the amount of the component of interest in cells of the cellular population of the sample based on the amount of marker bound to the component of interest.
 45. The method of claim 44, wherein said sample holder comprises a sample holder selected from i) a sample holder comprising a cuvette comprising a sample chamber configured to hold a sample, at least a portion of said cuvette comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface, wherein said optically transmissive surface and said reflective surface are configured effective that light passing through the optically transmissive surface simultaneously provides both epi-illumination and trans-illumination to said sample in the sample chamber, where epi-illumination comprises light traveling from an illumination source to the sample without reflection at a surface of the optically transmissive material, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material, and ii) a sample holder comprising a cuvette comprising a sample chamber having an optically transmissive floor, said cuvette having an outer surface comprising at least one concave or convex structure configured to provide mechanical support to the cuvette.
 46. A method for focusing a microscope, comprising: a) mixing a sample containing an object for microscopic analysis with a reference particle having a known size, effective to generate a mixture containing the sample and reference particle; b) positioning the mixture of step a) into a light path of a microscope; c) exposing the mixture of step a) to a light beam configured to visualize the reference particle; and d) focusing the microscope based on the position of the reference particle within the mixture or based on the sharpness of the image of the reference particle.
 47. The method of claim 46, wherein said mixture containing the sample and a reference particle is held within a sample holder selected from i) a sample holder comprising a cuvette comprising a sample chamber configured to hold a sample, at least a portion of said cuvette comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface, wherein said optically transmissive surface and said reflective surface are configured effective that light passing through the optically transmissive surface simultaneously provides both epi-illumination and trans-illumination to said sample in the sample chamber, where epi-illumination comprises light traveling from an illumination source to the sample without reflection at a surface of the optically transmissive material, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material, and ii) a sample holder comprising a cuvette comprising a sample chamber having an optically transmissive floor, said cuvette having an outer surface comprising at least one concave or convex structure configured to provide mechanical support to the cuvette. 48-50. (canceled)
 51. A system for imaging a sample, the system comprising a sample vessel containing said sample, a stage having a sample vessel receiver with an optically transparent surface; a light source for illuminating formed components in the sample through the stage, wherein the sample vessel has an interface surface configured to engage the optically transparent surface of the sample vessel receiver whereby the interface surface conforms to the optically transparent surface without significant distortion of light passing through the interface surface.
 52. The system of claim 51, wherein the interface surface of the sample vessel is formed from a polymer material.
 53. The system of claim 51, wherein the interface surface of the sample vessel is formed of a material softer than a material used to form the optically transparent surface of the sample vessel receiver.
 54. The system of claim 51, further comprising a compression unit for applying pressure to conform the interface surface to a shape configured to conform with the optically transparent surface of the sample vessel receiver.
 55. The system of claim 51, further comprising a handling unit configured to be coupled to the sample vessel to facilitate transport of sample vessel on and off the stage, and increase mechanical rigidity of the sample vessel.
 56. The system of claim 51, further comprising an opaque handling unit configured to be coupled to the sample vessel.
 57. The system of claim 51, where all imaging of the sample without passing light in a substantially straight line through one surface and out an opposing surface to a detector.
 58. The system of claim 51, where the light source is not located on one side of the sample vessel to deliver light to a detector on an opposite side of the sample vessel. 59-64. (canceled) 